Voluntary Report - public distribution

**Date:** 6/8/2006

**GAIN Report Number:** KS6064

KS6064

**Korea, Republic of**

**Biotechnology**

**Korean Testing Methods for GM Corn, Soybeans, and Potatoes**

**2006**

**Approved by:**

![](media/image1.wmf)Susan Phillips

U.S. Embassy

**Prepared by:**

Chris Frederick / Seung Ah Chung

**Report Highlights:**

The Korea Food & Drug Administration (KFDA) provided a translation of
Korea\'s official testing methods for biotech corn, soybeans, and
potatoes. This report also provides recommended information that foreign
laboratories include on their testing certificates for products exported
to Korea.

Includes PSD Changes: No

Includes Trade Matrix: No

Unscheduled Report

Seoul \[KS1\]

\[KS\]

Currently the Korea Food & Drug Administration (KFDA) has accredited
four Korean domestic laboratories for GMO testing. No laboratories in
foreign countries have been accredited so far. Beginning December 1,
2005, KFDA required accredited domestic laboratories to submit testing
certificates that contain information on the 17 items listed below. KFDA
would like foreign laboratories to complete the tests when issuing
certificates for products destined for Korea, even if the laboratories
are not accredited. To facilitate compliance KFDA has provided a
translation of its testing methods and requirements.

The accreditation of local laboratories for GMO testing does not change
the terms of enforcement of GMO labeling requirements. KFDA requires
either full IP documents, a government issued certificate, or a
notarized self-certification (for US origin products only) when
determining the exemption from GMO labeling. Importers and exporters can
submit testing certificates issued by either Korean or foreign
laboratories (government laboratories or private laboratories) for
products that do not contain foreign protein or DNA. However, if a
product tests positive for a GMO component during KFDA's random testing,
it will require a label stating that the product is GMO regardless of
what is stated on the original testing certificates. Testing
certificates issued by accredited laboratories do not guarantee that
products are exempt from random inspection.

KFDA believes accrediting local laboratories for GMO testing will
improve the credibility of the testing results but will not replace
KFDA's random testing.

The 17 items that will be included on the testing certificates are as
follows:

1.  Sample name (Korean, English)

2.  Manufacturer and importer (or client) name

3.  Production date and lot number of the product

4.  Sample weight submitted (kg)

5.  Sample packing appearance

6.  Sample description

7.  Dates test started and completed

8.  DNA extraction reagent or DNA extraction kit (e.g. Qiagen kit, CTAB
    method, etc.)

9.  Sample weight (g) used per DNA extraction and repetition number of
    DNA extraction

10. Concentration (ng/ul) and purity (A~260~/A~280~, A~260~/A~230~) of
    Extracted DNA and Elution volume (ul)

11. Accredited method or reference used for PCR (e.g. kit or primer
    name)

12. Target DNA region for PCR (e.g. Lectin, P35S, etc.)

13. Repetition number for PCR

14. Limit of Detection (LOD)

15. Concentration (ng/ul) and Quantity (ng) of Extracted DNA used per
    PCR

16. Electrophoresis picture images of PCR products (positive and
    negative controls should be included)

17. Final test result (Detected, Not detected, Indeterminate)

Note: The translation below is provided by KFDA. However, if there is
any discrepancy between the English translation and the original Korean
text, the Korean texts shall prevail.

**Korean Food Code**

# Chapter 7. Testing Methods in General

**23. Testing methods of GM foods**

Testing methods of genetically modified foods involve recombinant genes
and foreign proteins derived from such genes, and refer to polymerase
chain reaction (PCR) analysis for recombinant genes, and immunoassay
based on antigen-antibody reaction for foreign proteins.

For agricultural products such as grains and pulses and slightly
processed agricultural products made by simply crushing agricultural
products, both analytical methods for recombinant genes and foreign
proteins may be used while the analytical methods for recombinant genes,
not analytical methods for proteins, are used for processed foods due to
denaturing, decomposition, of proteins during manufacturing/processing.
For the analysis of foreign proteins, commercial test kits using
enzyme-linked immunosorbent assay (ELISA) techniques or lateral flow
strips may be used.

The qualitative analysis of recombinant genes uses standard PCR
equipment while the quantitative analysis of the genes uses real-time
PCR equipment.

However, both qualitative and quantitative analytical methods are
applicable to agricultural products and their merely ground products
while only the qualitative analysis is applied to processed foods until
the quantitative method is developed and established.

**1) Pre-treatment of samples**

\(1\) Washing and grinding of grains/pulses

Take at least 3,000 kernels or more than 1 kg of a grain or pulse sample
and homogeneously grind them using a grinder. Such ground powder is used
for analysis. If the sample contains chemicals or other extraneous
materials that may affect test results, put the sample in 1% SDS (Sodium
Dodecyl Sulfate) solution and stir it well until bubbles are
sufficiently formed. After washing, remove the solution and rinse the
sample with distilled water more than 10 times until such bubbles
disappear. Dry the sample and use it for analysis.

(2) Pre-treatment of processed foods with high water content

For processed foods with high water content, such as soybean milk,
homogeneously grind solid materials obtained from centrifugation at
8,000xg for 15 minutes or from overnight drying at 55 °C or evaporation
of water using a freeze dryer and use such ground powder for analysis.

(3) Pre-treatment of processed foods with high sugar content

For processed foods with high sugar content, such as sweet stuffs coated
with syrup, grind them with a grinder, add distilled water to dissolve
the sugar, perform centrifugation, and take the solid portion. Repeat
this procedure until sugar is sufficiently removed. Use the resultant
solid material for analysis.

(4) Pre-treatment of processed foods with high absorptive property

For processed foods with high absorptive property, add appropriate
quantity of sterilized distilled water, homogenize them with a grinder,
perform centrifugation at 8,000xg for 15 minutes at room temperature,
and take the solid material for analysis.

**2) Weighing of samples and prevention of contamination**

To weigh samples for extraction of DNA, clean a precision scale and its
neighboring area with 70% ethanol to prevent contamination. Then, place
the plastic wrap generally used for food packaging over the scale and
conduct weighing of samples in tubes to be used in extraction of DNA. If
possible, use disposable materials for weighing. Otherwise, use
different ones for different samples. After weighing, clean the tube
mouth and close the tube.

**3) Extraction and purification of DNA**

Extraction and purification of DNA can be conducted by the CTAB method
(extraction and purification of DNA using a mixture of a surfactant,
Cetyl Trimethyl Ammonium Bromide (CTAB), and phenol/chloroform) or the
method using the DNA extraction kit in silica gel membrane type,
silica-based resin type, or magnet-adsorptive bead type.

The CTAB method is applicable to various areas, leaves virtually no PCR
inhibitors, and provides high-purity DNA. However, currently marketed
DNA extraction kits have limited application areas, so their
applicability to a specific type of processed foods has to be separately
evaluated.

With use of such methods, DNA can be extracted from soybeans, corns,
potatoes, or their processed products, and purified for PCR analysis.
Conduct the DNA extraction process two times and perform PCR analysis
for each DNA extract.

Distilled water in this test method means high-purity water of 17 MΩ/cm
produced through reverse osmosis membrane or others, unless otherwise
specified. Such distilled water should not be contaminated with DNA or
DNase.

(1) CTAB method

Place 2g of the ground sample in a polypropylene tube (50ml), add 15ml
of CTAB buffer, and homogenize the mixture using a vortex mixer. Add
30ml of CTAB buffer and leave it at 55 °C for 30 minutes. Take 600μl of
the mixture sufficiently mixed and homogenized into 1.5ml tube, add
500μl of phenol/chloroform mixture into the tube, mix the solution well,
and centrifuge it at 7,500xg for 15 minutes at room temperature.
Transfer the supernatant to a new tube (1.5ml), add 500μl of
chloroform/isoamyl alcohol mixture, mix it well using a mixer, and
centrifuge it at 7,500xg for 15 minutes at room temperature. Transfer
the supernatant to a new tube (1.5ml), add the same quantity of
isopropyl alcohol, mix the solution by overturning the tube (about 10
times), and centrifuge it at 7,500xg for 10 minutes at room temperature.
Discard the clear supernatant. Gently add 500μl of 70% ethanol along the
wall of the tube to the pellet, centrifuge it at 7,500xg for 1 minute at
room temperature, and suck out as much of the supernatant using a
micropipette as possible without touching the pellet. Then, dry the
pellet, being careful not to dry it completely. Add 50μl of TE buffer
(pH 8.0), mix the solution well, and leave it at room temperature for 15
minutes for complete dissolution while overturning it occasionally. (If
it is not dissolved well, leave it at 4 °C for 12 to 24 hours for
complete dissolution.) To purify such DNA extract, add 5μl of RNase A
(10mg/ml) and leave the mixture at 37 °C for 30 minutes. After adding
200μl of CTAB buffer, add 250μl of chloroform-isoamyl alcohol mixture,
mix the solution using a mixer, centrifuge it at 7,500xg for 15 minutes
at room temperature, and transfer the supernatant to a new 1.5ml tube.
During this process, be careful not to disturb the medium layer. Add
200μl of isopropyl alcohol, mix the solution by overturning the tube
about 10 times, and centrifuge it at 7,500xg for 10 minutes at room
temperature. Discard the supernatant using a micropipette without
touching the pellet. Add 200μl of 70% ethanol along the wall of the tube
to the pellet and then, remove the supernatant using a micropipette.
Centrifuge it at 7,500xg for 1 minute at room temperature, suck out as
much ethanol as possible using a micropipette, and dry the pellet, being
careful not to dry it completely. Add 50 μl of sterilized distilled
water or TE buffer (pH 8.0) to the dried pellet and leave it at room
temperature for 15 minutes. During this process, overturn the tube
occasionally to dissolve it completely. Use the solution as the DNA
sample stock solution. (If it is not dissolved well, leave it at 4 °C
for 12 to 24 hours for complete dissolution.)

\[Preparation of solutions\]

 CTAB buffer

Place 8 ml of 0.5M EDTA (Ethylene Diamine Tetra Acetic Acid) solution
(pH 8.0), 20ml of 1M Tris/HCl buffer (pH 8.0), and 56ml of 5M salt
solution in a beaker, add enough distilled water to bring the volume to
approximately 150ml, add 4g of cetyl trimethyl ammonium bromide (CTAB)
while stirring, and dissolve it completely. Add enough distilled water
to bring the total volume to 200ml and autoclave the solution at 121 °C
for 15 minutes.

 Phenol/chloroform mixture

Mix phenol saturated with 1M Tris/HCl solution (pH 8.0), chloroform, and
isoamyl alcohol at 25:24:1 (v/v/v).

 Chloroform/isoamyl alcohol mixture

Mix chloroform and isoamyl alcohol at 24:1 (v/v).

 TE buffer (pH 8.0)

Prepare a solution having final concentrations of 10 mM Tris/HCl (pH
8.0) and 1 mM EDTA (pH 8.0) with sterilized distilled water.

(2) Silica-gel membrane-type kit method

Extract DNA using QIAGEN Plant Maxi kit or other equivalent kits
according to the manufacturer\'s instructions.

 Soybeans or processed soybean products

When QIAGEN Plant Maxi kit is used, place 1g of uniformly ground sample
in a polypropylene tube (50ml), add 10ml of AP1 buffer previously warmed
at 65 °C and 20μl of RNase A (100 mg/ml), mix them well using a mixer,
and leave the mixture at 65 °C for 1 hour. During this process, use a
mixer for 10 seconds at the 15-minute intervals for complete mixture.
After 1-hour reaction, centrifuge it at 3,000xg for 10 minutes at room
temperature and take the supernatant (7ml) into a new 50ml tube, being
careful not to touch the pellet and the membrane formed. Add 2.5ml of
AP2 buffer, leave the mixture in ice for 15 minutes, and centrifuge it
at 3,000xg for 35 minutes at room temperature. Then, transfer the clear
supernatant (8ml) to a QIA Shredder Spin Column, and perform
centrifugation at 3,000xg for 5 minutes at room temperature using a
swing rotor. Transfer the elute (7.5ml) into a new 50ml tube, mix it
well for 10 seconds, and place the solution (6.8ml) in a new 50ml tube.
Add 1.5 times the elute volume of AP3 buffer/ethanol mixture (10.2 ml),
mix it well for 10 seconds, apply it to a DNeasy Maxi Spin Column, and
perform centrifuge at 3,000xg for 5 minutes at room temperature using a
swing rotor to have DNA attached to the column. Discard the elute. Apply
12ml of AW buffer to the column, perform centrifugation at 3,000xg for
15 minutes at room temperature, and conduct cleaning. Transfer the
column into a new 50ml tube, add 1ml of sterilized distilled water
previously warmed at 65 °C, leave it at room temperature for 5 minutes,
and perform centrifugation at 3,000xg for 10 minutes at room temperature
to elute DNA. Take the elute into 2ml tube, add the same quantity of
isopropyl alcohol, overturn it 10 times, and leave it at room
temperature for 5 minutes. Perform centrifugation at 12,000xg for 15
minutes at 4 °C and remove the supernatant using a micropipette, being
careful not to touch the pellet. Add 500μl of 70% ethanol and gently
dissolve the pellet attached to the wall of the tube. Perform
centrifugation at 12,000xg for 3 minutes at 4 °C, remove the remaining
ethanol using a micropipette, and dry the precipitate. Add 50μl of
sterilized distilled water or TE buffer (pH 8.0) to the dried
precipitate and leave it at 4 °C for 12 to 24 hours for complete
dissolution. Use this as the DNA sample stock solution.

 Corns or processed corn products

When QIAGEN Plant Maxi kit is used, place 1g of uniformly ground sample
in a polypropylene tube (50ml), add 5ml of AP1 buffer previously warmed
at 65 °C and 10μl of RNase A (100mg/ml), mix them well using a mixer,
and leave the mixture at 65 °C for 1 hour. During this process, use a
mixer for 10 seconds at the 15-minute intervals for complete mixture.
Add 1.8ml of AP2 buffer, leave the mixture on ice for 15 minutes, and
centrifuge it at 3,000xg for 15 minutes at room temperature. Then,
transfer the clear supernatant (4.2ml) to a QIA Shredder Spin Column,
and perform centrifugation at 3,000xg for 5 minutes at room temperature
using a swing rotor. Transfer the elute (4ml) into a new 50ml tube, mix
it well for 10 seconds, and place the solution (3.4ml) in a new 50ml
tube. Add 1.5 times the elute volume of AP3 buffer/ethanol mixture
(5.1ml), mix it well for 10 seconds, apply it to a DNeasy Maxi Spin
Column, and perform centrifuge at 3,000xg for 5 minutes at room
temperature using a swing rotor to have DNA attached to the column.
Discard the elute. Apply 12ml of AW buffer to the column, perform
centrifugation at 3,000xg for 15 minutes at room temperature, and
conduct cleaning. Transfer the column into a new 50ml tube, add 1ml of
sterilized distilled water previously warmed at 65 °C, leave it at room
temperature for 5 minutes, and perform centrifugation at 3,000xg for 10
minutes at room temperature to elute DNA. Take the elute into 2ml tube,
add the same quantity of isopropyl alcohol, overturn it 10 times, and
leave it at room temperature for 5 minutes. Perform centrifugation at
12,000xg for 15 minutes at 4 °C and remove the supernatant using a
micropipette, being careful not to touch the pellet. Add 500μl of 70%
ethanol and gently dissolve the pellet attached to the wall of the tube.
Perform centrifugation at 12,000xg for 3 minutes at 4 °C, remove the
remaining ethanol using a micropipette, and dry the precipitate. Add
50μl of sterilized distilled water or TE buffer (pH 8.0) to the dried
precipitate and leave it at 4 °C for 12 to 24 hours for complete
dissolution. Use this as the DNA sample stock solution.

 Potatoes and processed potato products

When QIAGEN DNeasy Plant Mini kit is used, place 200mg of uniformly
ground sample in a centrifuge tube (15ml), add 1.5ml of AP1 buffer
previously warmed at 65 °C and 10μl of RNase A (100mg/ml), mix it well
using a mixer, and leave the mixture at 65 °C for 15 minutes. During
this process, use a mixer for 10 seconds at the 5-minute intervals for
complete mixture. Add 400 μl of AP2 buffer, leave the mixture in ice for
5 minutes, and centrifuge it at 10,000xg for 5 minutes at room
temperature. Then, transfer the clear supernatant to another centrifuge
tube, add 500μl of the supernatant to a QIA Shredder Spin Column, and
perform centrifugation at 10,000xg for 2 minutes at room temperature.
Transfer the elute into a new tube. Repeat this process for the
remaining supernatant. Transfer 2ml of the elute to two 2ml tubes, add
1.5 times the elute volume of AP3 buffer/ethanol mixture, and mix them
well for 10 seconds. Take 500 μl of the mixture into a DNeasy Mini Spin
Column and perform centrifuge at 10,000xg for 1 minute at room
temperature to have DNA attached to the column. Discard the elute. Apply
500μl of AW buffer to the column, perform centrifugation at 10,000xg for
1 minute at room temperature for cleaning. Repeat this process after
addition of 500 μl of AW buffer. To dry the column, perform
centrifugation at 10,000xg for 15 minutes at room temperature and
transfer to a new 1.5ml tube. Add 50 μl of sterilized distilled water
previously warmed at 65 °C, leave it at room temperature for 5 minutes,
and perform centrifugation at 10,000xg for 1 minute at room temperature
to elute DNA. Once again, add 50 μl of sterilized distilled water
previously warmed at 65 °C and repeat this process. Use this as the DNA
sample stock solution.

**Note**

AP1 buffer, AP2 buffer, and RNase A used in QIAGEN Plant Maxi kit and
QIAGEN DNeasy Plant Mini kit for the silica-gel membrane-type kit method
for extraction and purification of DNA are separately sold.

**4) Determination of the purity and concentration of DNA in DNA sample
stock solution and its concentration**

Appropriately dilute the DNA sample stock solution with TE buffer (pH
8.0) and determine the absorbance (A) of such dilutions at 260 nm using
a spectrophotometer. Use the value of 1 as 50ng/μl DNA. Meanwhile,
measure the absorbance at 230 nm, 260 nm, and 280 nm to determine the
purity of DNA extract. If the ratios of A~260~/A~280~ and A~260~/A~230~
are within the range of 1.7 to 2.0, the DNA is fully purified and
suitable for PCR. However, such purity criteria cannot be applicable to
processed foods. If the ratio of A~260~/A~280~ is low and there may be
contamination of protein impurities, treat the sample with protease and
recover DNA. If the ratio of A~260~/A~230~ is low, treat the sample with
amylase and recover DNA for use in PCR.

Based on the determined DNA concentration of the DNA sample stock
solution, dilute the DNA stock solution with TE buffer (pH 8.0) or
sterilized distilled water to a concentration required for subsequent
PCR reaction, mix it well, dispense the solution in 20 μl portions into
0.5ml tubes, and store them at below --20 °C for use as the DNA sample
solution. Use the DNA sample solution for PCR reaction immediately after
melting at room temperature and mixing it well. Any remaining sample
solution after PCR reaction should not be used and should be discarded.
Unless the concentration of the DNA sample stock solution reaches that
required for PCR reaction, conduct extraction again. If the
concentration is lower than that required for PCR even after
re-extraction, use it as the DNA sample solution without dilution or
conduct the following concentration process.

For concentration of DNA sample stock solution, add 1/10 times the DNA
solution of 3M sodium acetate (pH 5.2) (1/5 times the DNA solution of
10M ammonium acetate), add 2 times the solution of cold ethanol, and
perform centrifugation at 12,000xg for 15 minutes. (If isopropyl alcohol
is used, add 1 time the solution and perform centrifugation at room
temperature.) Discard the supernatant, clean the pellet with 70% ethanol
to remove salts, dissolve it with appropriate volume of TE buffer (pH
8.0) or sterilized distilled water, and use the solution.

    

If the initial DNA sample stock solution is a small quantity, add
sterilized distilled water to make 300 - 400μl and perform
concentration.

**5) Qualitative analysis method**

Qualitative analysis is conducted with use of general PCR method. DNA
extract is used as template DNA and amplified by use of primers to
detect the amplified DNA by electrophoresis.

    

\"In-house\" primers or GMO detection kit for PCR reaction to detect GM
foods can be used. As a very small amount of template DNA is amplified
in PCR reaction, it is very important to avoid contamination of DNA
other than target DNA (particularly PCR byproducts). Therefore, careful
attention should be paid to devices, instruments, reagents, and
environmental conditions. Further, DNase, secreted from the human skin
surface, should be prevented from entering the PCR reaction mixture
because it decomposes DNA. Therefore, disposable tubes and tips should
be autoclaved at 121 °C for more than 20 minutes prior to use. All kinds
of solutions to be used in preparation of various test solutions, except
those vulnerable to heat, should be autoclaved at 121 °C for more than
15 minutes prior to use.

Contamination of sterilized distilled water or TE buffer (pH 8.0) with
DNase will trigger the negative effects. Therefore, it is recommended
that each technician prepare them for immediate use as necessary. When
handing DNA, use an isolated space, such as clean bench. In addition,
clean the workstation with 70% ethanol and wear non-powdered rubber
gloves.

(1) Preparation of primers

 How to use \"in-house\" primers

Primers for test of GM soybeans, corns, or potatoes can be synthesized
according to the method published in the AOAC Journal as shown in
Table 1. Nippon Gene (Japan) makes and sells such primers (Table 2).
Primers that will produce the equivalent results can be designed,
prepared, and used.

 How to use commercial GMO detection kit

Commercial kits for test of GM soybeans, corns, or potatoes can be used.
Such kits should have the performance equivalent to the method published
in the AOAC Journal (see Table 1). In addition, primers that generate
more than 200 bp of PCR byproducts are not suitable for analysis of
processed foods.

Table 1. Primers and probes used in the method published in the AOAC
journal.

+--------+--------+-----------+---------------------------------------+
| P      | Pr     | Primers/  | Base Sequences                        |
| urpose | oducts |           |                                       |
|        |        | Probes    |                                       |
+--------+--------+-----------+---------------------------------------+
| In     | So     | L         | 5\'-GCC CTC TAC TCC ACC CCC A-3\'     |
| ternal | ybeans | e1n02-5\' |                                       |
| st     |        |           | 5\'-GCC CAT CTG CAA GCC TTT TT-3\'    |
| andard | Corns  | L         |                                       |
| genes  |        | e1n02-3\' | 5\'-FAM-AGC TTC GCC GCT TCC TTC AAC   |
|        |        |           | TTC AC-TAMRA-3\'                      |
|        |        | Le1-Taq   |                                       |
|        |        |           | 5\'-CTC CCA ATC CTT TGA CAT CTG C-3\' |
|        |        |           |                                       |
|        |        |           | 5\'-TCG ATT TCT CTC TTG GTG ACA       |
|        |        | SSllb     | GG-3\'                                |
|        |        | 1-5\'     |                                       |
|        |        |           | 5\'-FAM-AGC AAA GTC AGA GCG CTG CAA   |
|        |        | SSllb     | TGC A-TAMRA-3\'                       |
|        |        | 1-3\'     |                                       |
|        |        |           |                                       |
|        |        | SSllb-Taq |                                       |
|        |        |           |                                       |
|        |        |           |                                       |
+--------+--------+-----------+---------------------------------------+
| Scr    | CMV    | P35S      | 5\'-ATT GAT GTG ATA TCT CCA CTG ACG   |
| eening | p35S   | 1-5\'     | T-3\'                                 |
|        |        |           |                                       |
|        | tNOS   | P35S      | 5\'-CCT CTC CAA ATG AAA TGA ACT TCC   |
|        |        | 1-3\'     | T-3\'                                 |
|        |        |           |                                       |
|        |        | P35S-Taq  | 5\'-FAM-CCC ACT ATC CTT CGC AAG ACC   |
|        |        |           | CTT CCT-TAMRA-3\'                     |
|        |        |           |                                       |
|        |        |           | 5\'-GTC TTG CGA TGA TTA TCA TAT AAT   |
|        |        | NOS ter   | TTC TG-3\'                            |
|        |        | 2-5\'     |                                       |
|        |        |           | 5\'-CGC TAT ATT TTG TTT TCT ATC GCG   |
|        |        |           | T-3\'                                 |
|        |        |           |                                       |
|        |        | NOS ter   | 5\'-FAM-AGA TGG GTT TTT ATG ATT AGA   |
|        |        | 2-3\'     | GTC CCG CAA-TAMRA-3\'                 |
|        |        |           |                                       |
|        |        | NOS-Taq   |                                       |
|        |        |           |                                       |
|        |        |           |                                       |
+--------+--------+-----------+---------------------------------------+
| Stru   | \[Soyb |           |                                       |
| ctural | eans\] |           |                                       |
| genes  |        | RRS       | 5\'-CCT TTA GGA TTT CAG CAT CAG       |
|        | RRS    | 01-5\'    | TGG-3\'                               |
|        |        |           |                                       |
|        | \[C    | RRS       | 5\'-GAC TTG TCG CCG GGA ATG-3\'       |
|        | orns\] | 01-3\'    |                                       |
|        |        |           | 5\'-FAM-CGC AAC CGC CCG CAA ATC       |
|        | Bt176  | RRS-Taq   | C-TAMRA -3\'                          |
|        |        |           |                                       |
|        | Bt11   |           |                                       |
|        |        |           |                                       |
|        | GA21   |           | 5\'-TGT TCA CCA GCA GCA ACC AG-3\'    |
|        |        |           |                                       |
|        | T25    | Bt176     | 5\'-ACT CCA CTT TGT GCA GAA CAG ATC   |
|        |        | 2-5\'     | T-3\'                                 |
|        | MON810 |           |                                       |
|        |        | Bt176     | 5\'-FAM-CCG ACG TGA CCG ACT ACC ACA   |
|        |        | 2-3\'     |                                       |
|        |        |           | TCG A-TAMRA-3\'                       |
|        |        | Bt176-Taq |                                       |
|        |        |           | 5\'-AAA AGA CCA CAA CAA GCC GC-3\'    |
|        |        |           |                                       |
|        |        |           | 5\'-CAA TGC GTT CTC CAC CAA GTA       |
|        |        | Bt11      | CT-3\'                                |
|        |        | 3-5\'     |                                       |
|        |        |           | 5\'-FAM-CGA CCA TGG ACA ACA ACC CAA   |
|        |        | Bt11      | ACA TCA-TAMRA-3\'                     |
|        |        | 3-3\'     |                                       |
|        |        |           | 5\'-GAA GCC TCG GCA ACG TCA-3\'       |
|        |        | Bt11-Taq  |                                       |
|        |        |           | 5\'-ATC CGG TTG GAA AGC GAC TT-3\'    |
|        |        |           |                                       |
|        |        |           | 5\'-FAM-AAG GAT CCG GTG CAT GGC CG -  |
|        |        | GA21      | TAMRA-3\'                             |
|        |        | 3-5\'     |                                       |
|        |        |           | 5\'-GCC AGT TAG GCC AGT TAC CCA-3\'   |
|        |        | GA21      |                                       |
|        |        | 3-3\'     | 5\'-TGA GCG AAA CCC TAT AAG AAC       |
|        |        |           | CCT-3\'                               |
|        |        | GA21-Taq  |                                       |
|        |        |           | 5\'-FAM-TGC AGG CAT GCC CGC TGA AAT   |
|        |        |           | C- TAMRA-3\'                          |
|        |        |           |                                       |
|        |        | T25 1-5\' | 5\'-GAT GCC TTC TCC CTA GTG TTG A-3\' |
|        |        |           |                                       |
|        |        | T25 1-3\' | 5\'-GGA TGC ACT CGT TGA TGT TTG-3\'   |
|        |        |           |                                       |
|        |        | T25-Taq   | 5\'-FAM-AGA TAC CAA GCG GCC ATG GAC   |
|        |        |           | AAC AA-TAMRA-3\'                      |
|        |        |           |                                       |
|        |        |           |                                       |
|        |        | M810      |                                       |
|        |        | 2-5\'     |                                       |
|        |        |           |                                       |
|        |        | M810      |                                       |
|        |        | 2-3\'     |                                       |
|        |        |           |                                       |
|        |        | M810-Taq  |                                       |
|        |        |           |                                       |
|        |        |           |                                       |
+--------+--------+-----------+---------------------------------------+

Table 2. Primers prepared and sold by Nippon Gene (Japan)

+-----------------------+-----------+-----+----------------------------+
| Target Genes          | Primer    | A   | Remarks                    |
|                       | pairs     | mp. |                            |
|                       |           | s   |                            |
|                       |           | ize |                            |
+-----------------------+-----------+-----+----------------------------+
| \[Soybeans\]          |           |     |                            |
|                       |           |     |                            |
| Internal standard     | L         | 11  | *Le1*/sense                |
| genes                 | e1n02-5\' | 8bp |                            |
|                       |           |     | *Le1*/antisense            |
|                       | L         |     |                            |
|                       | e1n02-3\' |     | P35S/sense                 |
| Recombinant genes     |           | 10  |                            |
|                       | P35S1-5\' | 1bp | P35S-pro/antisense         |
| (transcription        |           |     |                            |
| initiation)           | P35S1-3\' |     | tNOS/sense                 |
|                       |           |     |                            |
| Recombinant genes     | NOS ter   | 15  | tNOS/antisense             |
|                       | 2-5\'     | 1bp |                            |
| (transcription        |           |     | CTP4 from *P.              |
| termination)          | NOS ter   |     | hybrida*/sense             |
|                       | 2-3\'     |     |                            |
| Recombinant genes     |           | 12  | *EPSPS*/antisense          |
|                       | RRS       | 1bp |                            |
|    (structural genes) | 01-5\'    |     |                            |
|                       |           |     |                            |
|                       | RRS       |     |                            |
|                       | 01-3\'    |     |                            |
+-----------------------+-----------+-----+----------------------------+
| \[Corns\]             |           |     |                            |
|                       |           |     |                            |
| Internal standard     | SSIIb     | 15  | *zSSIIb*/sense             |
| genes                 | 1-5\'     | 1bp |                            |
|                       |           |     | *zSSIIb*/antisense         |
|                       | SSIIb     |     |                            |
|                       | 1-3\'     |     | *zSSIIb*/sense             |
| Internal standard     |           | 11  |                            |
| genes                 | SSIIb     | 4bp | *zSSIIb*/antisense         |
|                       | 3-5\'     |     |                            |
|                       |           |     |  P35S/sense                |
|                       | SSIIb     |     |                            |
| Recombinant genes     | 3-3\'     | 10  |  P35S/antisense            |
|                       |           | 1bp |                            |
| (transcription        |  P35S     |     | tNOS/sense                 |
| initiation)           | 1-5\'     |     |                            |
|                       |           |     | tNOS/antisense             |
| Recombinant genes     |  P35S     | 15  |                            |
|                       | 1-3\'     | 1bp | *cryIA(b)*/sense           |
| (transcription        |           |     |                            |
| termination)          | NOS ter   |     | PEPC#9 intron /antisense   |
|                       | 2-5\'     |     |                            |
| Recombinant genes     |           | 10  | *adh1*-1S/sense            |
|                       | NOS ter   | 0bp |                            |
| (Bt176 detection)     | 2-3\'     |     | *cryIA(b)*/antisense       |
|                       |           |     |                            |
| Recombinant genes     | Bt176     |     | OTP/sense                  |
|                       | 2-5\'     | 12  |                            |
| (Bt11 detection)      |           | 7bp | *m-epsps*/antisense        |
|                       | Bt176     |     |                            |
| Recombinant genes     | 2-3\'     |     | *pat*/sense                |
|                       |           |     |                            |
| (GA21 detection)      | Bt11      | 13  | t35S/antisense             |
|                       | 3-5\'     | 3bp |                            |
| Recombinant genes     |           |     | *hsp70* /sense             |
|                       | Bt11      |     |                            |
| (T25 detection)       | 3-3\'     |     | *cryIA(b)*/antisense       |
|                       |           | 149 |                            |
| Recombinant genes     | GA21      | bp  | CaM03/sense                |
|                       | 3-5\'     |     |                            |
| (M810 detection)      |           |     | CBH02/antisense            |
|                       | GA21      |     |                            |
| Recombinant genes     | 3-3\'     | 113 | *cry9C*/sense              |
|                       |           | bp  |                            |
| (CBH351 1st           | T25 1-5\' |     | t35S/antisense             |
| detection)            |           |     |                            |
|                       | T25 1-3\' |     |                            |
| Recombinant genes     |           | 17  |                            |
|                       | M810      | 0bp |                            |
| (CBH351 2nd           | 2-5\'     |     |                            |
| confirmation)         |           |     |                            |
|                       | M810      |     |                            |
|                       | 2-3\'     | 17  |                            |
|                       |           | 1bp |                            |
|                       | CaM03-5\' |     |                            |
|                       |           |     |                            |
|                       | CBH02-3\' |     |                            |
|                       |           |     |                            |
|                       | Cry9C-5\' |     |                            |
|                       |           |     |                            |
|                       | 3         |     |                            |
|                       | 5Ster-3\' |     |                            |
+-----------------------+-----------+-----+----------------------------+
| \[Potatoes\]          |           |     |                            |
|                       |           |     |                            |
| Internal standard     | Pss       | 21  | *S. tuberosum* sucrose     |
| genes                 | 01n-5\'   | 6bp | synthase/sense             |
|                       |           |     |                            |
|                       | Pss       |     | *S. tuberosum* sucrose     |
|                       | 01n-3\'   |     | synthase/antisense         |
| Internal standard     |           | 11  |                            |
| genes                 | UGP       | 1bp | UGPase/sense               |
|                       | ase01-5\' |     |                            |
|                       |           |     | UGPase/antisense           |
|                       | UGP       |     |                            |
| Recombinant genes     | ase01-5\' | 23  |  p-FMV/sense               |
|                       |           | 4bp |                            |
| (New Leaf Plus 1st    | p-        |     |  PLRV/antisense            |
| detection)            | FMV02-5\' |     |                            |
|                       |           |     | PLRV/sense                 |
| Recombinant genes     | P         | 17  |                            |
|                       | LRV01-3\' | 2bp | PLRV/antisense             |
| (New Leaf Plus 2nd    |           |     |                            |
| confirmation)         | PLRV      |     | p-FMV/sense                |
|                       | -rep1-5\' |     |                            |
| Recombinant genes     |           | 22  | PVY/antisense              |
|                       | PLRV      | 5bp |                            |
| (New Leaf Y 1st       | -rep1-3\' |     | PVY/sense                  |
| detection)            |           |     |                            |
|                       | p-        |     | PVY/antisense              |
|                       | FMV05-5\' | 16  |                            |
|                       |           | 1bp |                            |
| Recombinant genes     | PVY02-3\' |     |                            |
|                       |           |     |                            |
| (New Leaf Y 2nd       | pVY01-5\' |     |                            |
| confirmation)         |           |     |                            |
|                       | pVY01-3\' |     |                            |
+-----------------------+-----------+-----+----------------------------+

(2) Preparation of reagents for PCR

Taq DNA polymerase (or polymerase producing an equivalent result) and
other reagents should be prepared for PCR reaction according to the
following procedures. To confirm the successful amplification of gene
from the template (DNA extract), subject a mixture including a positive
control primer pair (to detect the internal standard gene) to PCR
reaction for each DNA sample solution.

 Preparation of standard DNA solution

Use the DNA solutions extracted from 0% and 2% standard sample (products
manufactured by IRMM and sold by Fluka or other equivalent products) as
the positive control. However, if PCR is conducted using primers from
the method published in the AOAC Journal (Table 1), the positive-control
plasmid prepared and sold by Nippon Gene can be used instead of DNA
solution extracted from 2% standard sample.

 Preparation of PCR reaction reagents

Reagents required for PCR reaction include A) Tag DNA polymerase
(heat-resistant DNA polymerase), B) dNTPs (deoxynucleotides solution),
C) PCR buffer (if MgCl~2~ containing Mg^+2^ is not included, MgCl~2~ is
separately required), and D) primers. If the primers from the method
published in the AOAC Journal (Table 1) are used, general composition of
PCR reaction solution is summarized in Table 3.

Table 3. Composition of PCR reaction solution

  -------------- --------- ------------------------------ -----------------
  Components     Stock     Final conc. (tube)             Volume (for 1
                 Soln.                                    reaction)

  DNA            5U/㎕     0.625U                         0.125㎕
  polymerase\*                                            

  Buffer         10 x      1×                             2.5㎕

  MgCl~2~\*\*    25mM      1.5mM                          1.5㎕

  dNTPs          2.5mM     200μM                          2㎕

  Primer         25μM,     0.5μM, each                    0.5㎕
                 each                                     

  Template DNA   20ng/㎕   50ng                           2.5㎕

  Sterilized DW            added to make the final volume 15.875㎕
                           of 25㎕                        

  Total                                                   25㎕
  -------------- --------- ------------------------------ -----------------

\*   For DNA polymerase, use Taq DNA polymerase that is used after
activation at 95 °C for 10 minutes.

\*\*  MgCl~2~ concentration greatly affects the specificity and output
of amplification. In general, 1.5 to 2.5mM is widely used. If Mg^2+^ is
in excess, non-specific PCR products are increased. If Mg^2+^ is
insufficient, PCR products are decreased.

 Preparation of controls

Negative controls include A) those not containing DNA (to determine the
contamination of PCR reaction system) and B) those not containing
primers (to determine the contamination of reaction system, except
primers). Positive controls include C) those containing non-GMO DNA (to
determine if extraction of DNA is correctly done, DNA extracted from 0%
standard sample taken from the same agricultural product (products
prepared by IRMM and sold by Fluka or other equivalent products)) and D)
those containing target recombinant DNA to be detected (to determine if
the PCR reaction system is correct, DNA extracted from 2% standard
sample (products prepared by IRMM and sold by Fluka or other equivalent
products)). Subject such negative and positive controls to PCR reaction
together with the sample.

(3) PCR

PCR reaction of each DNA extract is conducted in two confirmation tests
according to the following procedure. In the first confirmation test,
primers for internal standard gene and transcription initiation gene
(promoter 35S) and/or transcription termination gene (NOS terminator)
are used for PCR reaction. From this test, DNA extract that is found to
have both amplification products (internal standard gene and 35 S
promoter and/or NOS terminator) in more than one extracted solution out
of two extraction samples is used as template DNA for the second
confirmation test with primers for structural gene.

 Preparation of master mixture

Determine the number of tubes to be used under consideration of the
number of samples for PCR reaction and calculate the amount of master
mixture based on the resultant total volume and some extra volume.
Prepare the PCR reaction solution according to Table 3, except template
DNA. Handle the master mixture on ice and dispense 22.5 μl to each PCR
tube. For negative controls that do not use primers, take 22 μl of
master mixture not containing primers and add 0.5 μl of sterilized
distilled water.

 Addition of template DNA

Add 2.5μl of template DNA solution to 22.5μl of master mixture. Conduct
addition of template DNA in the order of negative controls, DNA extract
(20ng/μl), and positive controls.

 PCR amplification

Conduct PCR after dispensing all reaction solutions. Reaction conditions
for PCR depend on primers. Table 4 shows the reaction conditions when
primers prepared and sold by Nippon Gene are used. After starting the
first denaturation by keeping the mixture at 95 °C for 10 minutes,
perform denaturation at 95 °C for 30 seconds, annealing at 60 °C for 30
seconds, and extension at 72 °C for 30 seconds as one cycle. Repeat this
cycle 40 times. Then, perform final elongation at 72 °C for 7 minutes.
When all reactions are completed, keep the mixture at 4 °C. Conduct
electrophoresis to confirm the amplification results or store such PCR
products at freezing condition.

Table 4. PCR reaction conditions

+-------------------------+------------------+-----------+-------------+
|                         | Temperature      | Time      | Cycle       |
+-------------------------+------------------+-----------+-------------+
| First denaturation      | 95 °C            | 10 min.   | 1 cycle     |
+-------------------------+------------------+-----------+-------------+
| Denaturation            | 95 °C            | 30 sec.   | 40 cycle    |
|                         |                  |           |             |
| Annealing               | 60 °C            | 30 sec.   |             |
|                         |                  |           |             |
| Extension               | 72 °C            | 30 sec.   |             |
+-------------------------+------------------+-----------+-------------+
| Elongation              | 72 °C            | 7 min.    | 1 cycle     |
+-------------------------+------------------+-----------+-------------+
| Storage                 | 4 °C             | \-        | \-          |
+-------------------------+------------------+-----------+-------------+

＊ If necessary, the above conditions may be adjusted.

(4) Electrophoresis and judgment

The results from PCR amplification can be confirmed by electrophoresis
using agarose gel or polyacrylamide gel. The agarose gel electrophoresis
method is described here.

 Preparation of agarose gel

In this step, use of specially sterilized materials is not necessary,
but it is desirable to wear rubber gloves to prevent any possible risks,
such as contamination.

First, assemble the gel maker. The concentration of gel is determined
under consideration of the length of DNA to be electrophoresed and the
type of agarose gel. In this case, since DNA extracted from sample and
PCR-amplified products of about 100 to 200 bp are subjected to
electrophoresis, use the agarose gel suitable for this purpose according
to the manufacturer\'s instruction. Add TAE (Tris-acetate/EDTA) buffer
(0.5 x) to the required quantity of agarose, and heat the mixture to
dissolve the agarose. Further, since PCR-amplified products of below 200
bp are subjected to electrophoresis, it is desirable to use the agarose
of high gel strength. For the pre-staining method, cool the mixture to
approximately 55 °C when the agarose is sufficiently dissolved to result
in homogeneous gel, add 1μl ethidium bromide (EtBr) solution (10mg/ml)
per 100ml of the above solution, and mix it well. When the gel is cooled
down to about 55 °C, pour it into a gel maker, insert the comb, and
leave it for about 30 minutes to sufficiently solidify it. When the gel
is solidified, add TAE buffer (0.5 x) sufficiently to allow the gel to
be soaked in the buffer and remove the comb carefully. Install the
agarose gel in the electrophoresis bath and fill it with TAE buffer (0.5
x) enough to soak the upper side of the gel in the buffer.

 Electrophoresis

Add 1/6 x gel loading buffer to the PCR amplified reactant and mix them
well. Inject the mixture into the well of the gel carefully. To both end
wells, inject the appropriate marker DNA to determine the size of the
PCR amplified reactant. The volume should not exceed 10μl. After
injection, conduct electrophoresis at a constant voltage of 50 to 100 V
until the BPB (promophenol blue) in the gel loading buffer advances to
the 1/2 ～ 2/3 position of the gel. Then, stop the electrophoresis and
identify the electrophoretic pattern using the gel image analyzer.

 Staining of gel

For the post-staining method, transfer the electrophoresed gel to a
container containing 5μl of ethidium bromide (EtBr) per 100ml of TAE
buffer (0.5 x) (10mg/ml), place the container on a shaker, and perform
staining for 20 to 30 minutes while shaking the container gently. Then,
place the container in distilled water for about 30 minutes for
de-staining. When pre-staining is used, the procedures specified in this
section are not necessary. The electrophoresis results can be
immediately determined.

 

Note

Ethidium bromide (EtBr) is a luminescent reagent to be inserted into the
DNA double helix structure. It is a potent mutagenic material.
Therefore, careful attention should be paid. Be sure to wear rubber
gloves and mask when handling this reagent. It is desirable to use the
dedicated device for treatment of waste EtBr. Otherwise, treat EtBr with
activated charcoal and then, discard it. If necessary, use a specialized
company for disposal of highly concentrated EtBr.

 Gel image analysis

 Place the stained electrophoretic gel on a piece of plastic wrap spread
on the stage of a gel image analyzer or a trans-illuminator and radiate
ultraviolet rays. Identify the electrophoretic pattern using a CCD
camera and judge the presence of the target band by comparing it with
the marker DNA. Store the electrophoretic results as image data. When a
Polaroid camera is used, take a photo and store it.

(5)  Judgement of results

When the target PCR amplified band is found in the lane, judge the
results as follows:

 At first, PCR reactant should not be detected in negative controls (A
-  negative control not containing DNA and B - negative control not
containing primer). Then, for the positive control containing non-GMO
DNA (C), the PCR reactant for internal standard gene should be found,
but the PCR reactant for recombinant DNA should not be detected. In
addition, for the positive control containing target recombinant DNA,
both PCR reactants for internal standard gene and recombinant DNA should
be confirmed. If one of the above criteria is not met, conduct re-tests.

 If PCR analysis of two DNA extracts (from repeated extraction
processes) shows PCR reactants specific to internal standard gene and
recombinant DNA in more than one DNA extract through the first and
second confirmation tests, it is determined as \"detected\". If the PCR
reactant specific to internal standard gene is found and the PCR
reactant specific to recombinant DNA is not detected, it is determined
as \"not detected\". If the PCR reactant specific to internal standard
gene is not detected in both DNA extract samples, conduct re-test from
the process of DNA extraction. If the PCR amplified reactant specific to
internal standard gene is not found in the re-test, it is determined as
\"impossible\". (Refer to Table 5.)

 In the GMO plants, RRS, Bt11, Bt176, T25, and Mon810 use 35S as the
transcription initiation gene (promoter) and RRS, Bt11, and GA21 use NOS
as the transcription termination gene (terminator).

 If an agricultural product is judged as \"detected\" according to the
above qualitative analysis, it will be subjected to examination of
documents, including the classified distribution certificate, and
labeling. Further, the quantitative analysis will be conducted for the
relevant product in question. However, agricultural products unsuitable
for foods or exempted from safety assessment, such as StarLink corns
(CBH351), will not be subjected to such examination of documents, such
as the classified distribution certificate, and quantitative analysis.

**6) Quantitative analysis**

At present, the PCR-based quantitative analysis of foods produced by
recombinant DNA techniques dose not give the absolute quantity value.
However, it is possible to measure the relative quantity of recombinant
DNA present in such foods from the ratio of recombinant DNA to internal
standard gene according to the method published in the AOAC Journal.

\"Real-Time PCR\" method requires DNA probes having the base sequence
complimentary to template DNA and binding luminescent material as well
as primers for reaction of polymerase used in general PCR method. As
polymerase reaction continues, these come to specifically bind with the
increasing reactants and luminescent materials become separated from
probes. The amount of such separated luminescent materials are
dynamically measured and the results are used to calculate the amount of
template DNA.

    

Further, the quantitative PCR method includes the competitive PCR not
requiring any special devices and the method utilizing the standard
curve obtained using reference standard samples.

Table 5. Flow chart of judgment (example)

  ------------ ------------ -------------- --------- -------------- -------------
  Samples      DNA Extracts 1st                      2nd            GMO Judgement
                            confirmation             confirmation   

                            Internal       P35S,     Structural     
                            standard gene  tNOS      gene           

  Sample No. 1 Extraction 1 \+             \+        \+             Detected

               Extraction 2 \+             \+        \+             

  Sample No. 2 Extraction 1 \+             \+        \+             Detected

               Extraction 2 \+             \+        \-             

  Sample No. 3 Extraction 1 \+             \+        \+             Detected

               Extraction 2 \+             \-        /              

  Sample No. 4 Extraction 1 \+             \+        \+             Detected

               Extraction 2 \-             \-        /              

  Sample No. 5 Extraction 1 \+             \+        \-             Not detected

               Extraction 2 \+             \+        \-             

  Sample No. 6 Extraction 1 \+             \+        \-             Not detected

               Extraction 2 \+             \-        /              

  Sample No. 7 Extraction 1 \+             \+        \-             Not detected

               Extraction 2 \-             \-        /              

  Sample No. 8 Extraction 1 \+             \-        /              Not detected

               Extraction 2 \+             \-        /              

  Sample No. 9 Extraction 1 \+             \-        /              Not detected

               Extraction 2 \-             \-        /              

  Sample No.   Extraction 1 \-             /         /              Impossible
  10                                                                

               Extraction 2 \-             /         /              
  ------------ ------------ -------------- --------- -------------- -------------

 + : Specific band detected

 - : Specific band not detected

 / : Test not required

1.    Extraction is conducted two times for one sample to perform PCR
reaction. For Sample No. 10, one extraction is additionally conducted.

2.    Depending on the GM varieties, 35 S promoter or NOS terminator may
not be detected.

3.    If primers sold by Nippon Gene (Japan) are used for PCR test of
potatoes or StarLink corns (CBH351), use the primers for the first
detection instead of the promoter and the terminator.

(1) Real-time PCR

This quantitative PCR method using TaqMan Chemistry employs a
luminescent oligonucleotide probe, which anneals in the DNA sequence
between the both primer pairs (3\' and 5\') and has two dyes (reporter
and quencher). When this probe is hydrolyzed by the 5\'-nuclease
activity of the DNA polymerase, the reporter dye is separated from the
quencher dye and emits luminescence. The strength of luminescence shows
exponential increase for the PCR cycle. Therefore, by comparing the PCR
cycle number which has reached a certain luminescence, the amount of
original DNA can be obtained.

    

The quantitative analysis of GM products is to determine the amount of
recombinant gene compared to the internal standard gene which is the
universally present gene in the non-GMOs.

(2) Quantification of soybeans produced by recombinant DNA techniques

In the case of soybeans, use the lectin-gene that is ubiquitous in
soybeans as the internal standard gene. The number of copies of the
lectin-gene in the DNA sample is obtained by quantitative PCR which uses
a primer pair (Le1-n02) detecting the lectin gene and a probe (Le1-Taq)
detecting the recombinant gene. At the same time, the number of copies
of the recombinant gene in the sample is obtained by another
quantitative PCR using a primer pair (RRS-01) and probe (RRS-Taq). The
percentage of GMO in sample is obtained from the following way. Divide
the number of recombinant gene copies by the number of the lectin gene
copies. And divide this value again by the internal standard ratio
(which is determined previously), and multiply the result by 100.

(3) Quantification of corns produced by recombinant DNA techniques

In the case of corns, there are many GM varieties (Bt176, Bt11, T25,
Mon810, GA21) that have different target proteins. Therefore, quantify
each GM variety separately, based on the qualitative analysis results
and judge from the result of total of the quantitative value for each GM
variety.

    

The analysis method is similar to that for quantitative PCR of soybeans,
except the primer pair and probe to be used. In the case of corn, use
the starch synthase IIb (SSIIb) gene as the universally present internal
standard gene. Calculate the amount of GMO content using the number of
copies of starch synthase IIb (SSIIb) gene (obtained from the primer
pair SSIIb-3 and probe SSIIb-Taq) and the number of copies of target
recombinant gene (obtained from the primer pair and probe, which detects
target recombinant gene).

    

Bt176, Bt11, T25, MON810, and GA21 use Bt176-2 and Bt176-Taq, Bt11-3 and
Bt11-Taq, T25-1 and T25-Taq, M810-2 and M810-Taq, and GA21-3 and
GA21-Taq as primer pair and probe, respectively.

    

Internal standard ratios applicable to GM soybeans and corns depend on
the type of Real-Time PCR equipment and they are summarized in Table 6,
Table 7, Table 8, and Table 9.

Table 6. Quantitative internal standard ratios for GM soybeans and corns
(ABI PRISM^™^ 7700 & 5700)

+------+-------------------+-------------------+----------------------+
| Prod | Varieties         | Internal standard | Remarks              |
| ucts |                   | ratios            |                      |
+------+-------------------+-------------------+----------------------+
| Corn | 35S promoter      | 0.39              | SSIIb-3 and          |
|      | screening         |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | P35S-1 and P35S-Taq  |
+------+-------------------+-------------------+----------------------+
|      | Bt11              | 0.44              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | Bt11-3 and Bt11-Taq  |
+------+-------------------+-------------------+----------------------+
|      | GA21              | 2.01              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | GA21-3 and GA21-Taq  |
+------+-------------------+-------------------+----------------------+
|      | T25               | 0.34              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | T25-1 and T25-Taq    |
+------+-------------------+-------------------+----------------------+
|      | Bt176             | 1.99              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | Bt176-2 and          |
|      |                   |                   | Bt176-Taq            |
+------+-------------------+-------------------+----------------------+
|      | MON810            | 0.38              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | Mon810-2 and         |
|      |                   |                   | Mon810-Taq           |
+------+-------------------+-------------------+----------------------+
| Soy  | 40-3-2 (RRS)      | 1.05              | Le1-n02 and Le1-Taq  |
| bean |                   |                   |                      |
|      |                   |                   | RRS-01 and RRS-Taq   |
+------+-------------------+-------------------+----------------------+

Table 7. Quantitative internal standard ratios for GM soybeans and corns
(ABI PRISM^™^ 7900HT 96well)

+------+-------------------+-------------------+----------------------+
| Prod | Varieties         | Internal standard | Remarks              |
| ucts |                   | ratios            |                      |
+------+-------------------+-------------------+----------------------+
| Corn | 35S promoter      | 0.38              | SSIIb-3 and          |
|      | screening         |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | P35S-1 and P35S-Taq  |
+------+-------------------+-------------------+----------------------+
|      | Bt11              | 0.40              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | Bt11-3 and Bt11-Taq  |
+------+-------------------+-------------------+----------------------+
|      | GA21              | 1.99              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | GA21-3 and GA21-Taq  |
+------+-------------------+-------------------+----------------------+
|      | T25               | 0.34              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | T25-1 and T25-Taq    |
+------+-------------------+-------------------+----------------------+
|      | Bt176             | 2.02              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | Bt176-2 and          |
|      |                   |                   | Bt176-Taq            |
+------+-------------------+-------------------+----------------------+
|      | MON810            | 0.36              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | Mon810-2 and         |
|      |                   |                   | Mon810-Taq           |
+------+-------------------+-------------------+----------------------+
| Soy  | 40-3-2 (RRS)      | 1.04              | Le1-n02 and Le1-Taq  |
| bean |                   |                   |                      |
|      |                   |                   | RRS-01 and RRS-Taq   |
+------+-------------------+-------------------+----------------------+

Table 8. Quantitative internal standard ratios for GM soybeans and corns
(ABI PRISM^™^ 7900HT 384well)

+------+-------------------+-------------------+----------------------+
| Prod | Varieties         | Internal standard | Remarks              |
| ucts |                   | ratios            |                      |
+------+-------------------+-------------------+----------------------+
| Corn | 35S promoter      | 0.39              | SSIIb-3 and          |
|      | screening         |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | P35S-1 and P35S-Taq  |
+------+-------------------+-------------------+----------------------+
|      | Bt11              | 0.43              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | Bt11-3 and Bt11-Taq  |
+------+-------------------+-------------------+----------------------+
|      | GA21              | 2.06              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | GA21-3 and GA21-Taq  |
+------+-------------------+-------------------+----------------------+
|      | T25               | 0.37              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | T25-1 and T25-Taq    |
+------+-------------------+-------------------+----------------------+
|      | Bt176             | 2.12              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | Bt176-2 and          |
|      |                   |                   | Bt176-Taq            |
+------+-------------------+-------------------+----------------------+
|      | MON810            | 0.38              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | Mon810-2 and         |
|      |                   |                   | Mon810-Taq           |
+------+-------------------+-------------------+----------------------+
| Soy  | 40-3-2 (RRS)      | 1.00              | Le1-n02 and Le1-Taq  |
| bean |                   |                   |                      |
|      |                   |                   | RRS-01 and RRS-Taq   |
+------+-------------------+-------------------+----------------------+

Table 9. Quantitative internal standard ratios for GM soybeans and corns
(LightCycler)

+------+-------------------+-------------------+----------------------+
| Prod | Varieties         | Internal standard | Remarks              |
| ucts |                   | ratios            |                      |
+------+-------------------+-------------------+----------------------+
| Corn | 35S promoter      | 0.53              | SSIIb-3 and          |
|      | screening         |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | P35S-1 and P35S-Taq  |
+------+-------------------+-------------------+----------------------+
|      | Bt11              | 0.63              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | Bt11-3 and Bt11-Taq  |
+------+-------------------+-------------------+----------------------+
|      | GA21              | 2.63              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | GA21-3 and GA21-Taq  |
+------+-------------------+-------------------+----------------------+
|      | T25               | 0.31              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | T25-1 and T25-Taq    |
+------+-------------------+-------------------+----------------------+
|      | Bt176             | 2.60              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | Bt176-2 and          |
|      |                   |                   | Bt176-Taq            |
+------+-------------------+-------------------+----------------------+
|      | MON810            | 0.49              | SSIIb-3 and          |
|      |                   |                   | SSIIb-Taq            |
|      |                   |                   |                      |
|      |                   |                   | Mon810-2 and         |
|      |                   |                   | Mon810-Taq           |
+------+-------------------+-------------------+----------------------+
| Soy  | 40-3-2 (RRS)      | 1.01              | Le1-n02 and Le1-Taq  |
| bean |                   |                   |                      |
|      |                   |                   | RRS-01 and RRS-Taq   |
+------+-------------------+-------------------+----------------------+

(4) Standard solution for the standard curve

Prepare GM plasmid DNA solution of which the number of copies has
already been determined and use it as the standard solution. Dilute the
GM plasmid DNA solution with *Escherichia coli* ColE1/TE DNA solution (5
ng/μl) so as to make appropriate copy numbers for the standard curve
(e.g. 20, 125, 1,500, 20,000, and 250,000 copies per well).

(5) Preparation of the solution for the PCR reaction

Dissolve all reagents used in quantitative PCR at room temperature and
sufficiently mix them using a mixer. If the same tip is used
successively for dividing, the usual handling will not work from the
second time onward because the air is cooled in the pipette. It is
necessary to read up on how to use the pipette at low temperature in the
manual of the pipette before starting the procedure. Further, when
handling high viscous reagents, such as Universal PCR Master Mix, place
the pipette vertical to the solution surface, slowly push the button
until the feeling of stopping is firstly felt, push it deeper a lot,
relieve the button very slowly to suck Taq polymerase, push the button
again slowly to discharge the solution until the feeling of stopping is
firstly felt, and finally relieve the button very slowly to suck the
solution. On dispensing, place the pipette vertically, push the button
slowly to dispense the solution until the feeling of stopping is firstly
felt, remove the pipette from the tube, and then, discard the tip.

    

25μl solution for the PCR reaction contains 12.5μl of Universal PCR
Master Mix, 0.5μl of primer (25mM each), 0.5μl of probe (10mM), 9μl of
sterilized distilled water, 2.5μl of 20ng/μl DNA sample solution (50ng),
and 2.5μl of the standard solution for the standard curve or 2.5μl of
5ng/μl ColE1/TE buffer. 5ng/μl ColE1/TE buffer corrects the effect of
trace DNA being non-specifically absorbed onto the wall surface of the
tube and is used as negative control.

 Preparation of primer-probe mixture

Dilute primer and probe with sterilized distilled water to make the
final concentration of 1.25mM and 0.5mM, respectively, and mix them well
using a mixer.

 Preparation of Master Mix

Mix the primer-probe solution and Universal PCR Master Mix at the ratio
of 1:1.25 using a mixer. In order to minimize the differences between
PCR reactions, prepare PCR reaction solution for 3 wells per each DNA
sample solution. The volume of the Master Mix per each DNA sample should
be 81μl.

 Dispensing of Master Mix

Prepare 500μl tubes for template DNA (the number of samples), standard
solutions for the standard curve (5 per primer), and negative control
solutions (1 per primer), and number them. Dispense the above mix
solution of 78.75 μl into each 500μl tube.

 Addition of template DNA, standard solutions for the standard curve,
or negative control solutions into the above tubes containing master
mix.

To tubes containing master mix, add 8.75μl of template DNA (20ng/μl),
8.75μl of standard solutions for the standard curve, or 8.75μl of
negative control solutions. Use a mixer to mix them well. Dispense 25μl
of the mix solution to each well. Put the lid on the plate. At this
time, to be impartial, close alternately from both sides. Then, using a
special roller, seal up the wells. Lastly, observe the bottom of the
wells, and if there are bubbles, knock lightly a rim of the plate or
perform centrifugation (using the centrifuge for 96-well plate) to
remove bubbles.

(6) Quantitative PCR

Quantitative PCR is performed using the Real-Time PCR device. Set the
plate onto the device, input well data, check if the cover temperature
is around 105 °C, and begin the reaction. The reaction conditions are as
follows: After maintaining it at 50 °C for 2 minutes, keep it at 95 °C
for 10 minutes. After starting the reaction with a hot start method,
conduct 40 cycles of amplification reaction (30 seconds at 95 °C and 1
minute at 59 °C for 1 cycle). After the end of the reaction, analyze the
results.

(7) Preparation of the standard curve

The standard curve for the internal standard gene and the recombinant
gene can be obtained from the following way.

    

The following description is an example of preparing the standard curve
when ABI PRISM 7700 is used. If other types of equipment are used,
prepare the standard curve according to the relevant user\'s manual. On
the amplification curve plotting the increasing amount of luminescent
signals (Rn) against the number of reactions, select the Rn part where
luminescent signals from both of the standard solution for the standard
curve and DNA sample solution amplify exponentially, and draw a
threshold line (Th. line). At this time, the threshold line must not
cross the non-specific amplified curve from the negative control
solution (blank sample solution using ColE1 DNA solution). In addition,
determine Th. line through the following manipulation on the window of
\"Amplification Plot\".  Set 3 for Start and 15 for Stop of Baseline. 
Enter the value of 2^m^ to Mult\*Stddev. At first, m is 0 (m=0). (In
other words, enter the value of 1.)  Push \"Suggest\" button and then,
\"Update Calculations\" button.  Select \"Analysis\" and \"Standard
Curve\", check the correlation coefficient, slope, and Y-axis values of
the standard curve, and enter them into \"Th. line Decision Table\"
together with the value of Th. line. Increase the value of m by 1 and
repeat this procedure. If one of NTC amplification curves crosses Th.
line (when it is not possible to identify such cross by visual
examination, it is determined as \"crossed\" if Ct value is 40 (in the
case of 40-cycle PCR) in Experiment Report), write \"NTC\" in the
remark.  Repeat this procedure until the standard amplification curve
for the maximum number of copies used comes to cross Th. line.  If one
of standard amplification curves for the maximum number of copies
crosses Th. line, write \"plot out\" in the remark of \"Th. line
Decision Table\".  Calculate the amplification rate (A) and ｜ΔA｜, and
select Th. line satisfying the following conditions.

    

Definitions of A & ΔA: A= 10^(-1\ /slop)^, ΔA=(A~m+1~－A~m~)/A~m~×100 

    

Condition 1: Select Th. line at the midpoint in the section where ｜ΔA｜
of more than 2 sections is below 1% (min. n, max. m). However, if the
selected Th. line is not an integer, round the decimals. In this case,
the correlation coefficient of the standard curve obtained from the
selected Th. line should be above 0.99, the amplification rate (A)
should be below 2.1, and the selected Th. line should not cross the NTC
curve.

    

Condition 2: If the above Condition 1 is not met, adjust the acceptable
value of ｜ΔA｜ to 2%. If not met even after such adjustment, adjust the
acceptable value of ｜ΔA｜ to 3%, 4%, or 5%.

    

Condition 3: If there are several m values satisfying Condition 1 or
Condition 2, select the largest m value.

    

Retest: If it is not able to determine Th. line even when 5% is employed
for ｜ΔA｜, conduct re-test.

Use the point, where the Th. line and the luminescent signal of the
standard solution for the standard curve cross, as the \"threshold cycle
(Ct)\" and calculate the number of copies from Experiment Report. Next,
plot the Ct value for the logarithm of copies of the (each) standard
solutions for standard curve (x-axis). Use the approximate straight line
obtained from each Ct as the standard curve.

(8) Calculation of the content of the foods produced by recombinant DNA
techniques in samples

    

Obtain the Ct value for unknown DNA sample, by using the Th. line of the
standard curve. For each of the internal standard gene and recombinant
gene, calculate the numbers of original genes\' copies for all 3 wells
from the standard curve. Use the average of these values as the number
of copies of the original internal standard gene and original
recombinant gene. Calculate the amount of GMO contained in the products
from the following formula.

  -------------------- ------- -------------------------- ------- ---------------- ------- -----
  GMO content in       **=**   No. of copies (recombinant **×**   1                **×**   100
  products (%)                 gene)                                                       

                               No. of copies (internal            Internal                 
                               standard gene)                     standard ratio           
  -------------------- ------- -------------------------- ------- ---------------- ------- -----

    

In the case of soybeans, the relevant GM variety is only \"Roundup Ready
Soybean\", the herbicide-resistant one developed by Monsanto, and the
percentage of GMO in products can be calculated from the number of
copies of Le-1 and RRS genes.

(9) Judgement of results

For each DNA sample extracted two times, calculate total content of RRS,
GA21, Bt176, Bt11, T25, and MON810 from 6)-(2) and (3). If the average
is more than 3%, it is concluded that classified distribution has
possibly failed.

**  For information**

You can purchase GM standard plasmid DNA used in preparation of the
standard solution for the standard curve from Nippon Gene Co. (FASMARK
Co.) or Wako Co. In addition, you can purchase the primer pairs and
probes used in preparation of the solution for the PCR reaction from
those companies.

**7) Analysis of StarLink Corn (CHB351)**

Import and distribution of StarLink corn is prohibited in accordance
with Item No. 6 in Article 4 of the Food Sanitation Act. For raw corns
and ground corns, firstly conduct the screening process with use of any
commercially available kits (Trait Bt9 Lateral Flow Test kit, Cry9C
QuickStix kit, or others) according to the lateral flow strip method. If
the screening result is positive, conduct the PCR reaction for
confirmation. For other processed foods, use the PCR method to determine
the presence of GM corns.

(1) Lateral flow strip method

Perform the test according to the manufacturer\'s instruction.

(2) PCR method

 DNA extraction

Use DNA solution obtained from repeated extraction (2 times) according
to the method described in 3) Extraction and purification of DNA and
perform PCR reaction according to the qualitative PCR method described
in 5) Qualitative analysis method. Use the following primers.

 Primer for detection of CBH351

A.  Use SSIIb-1 primer to determine the internal standard gene.

B.  Primer for 1st detection of CBH351

     F-primer (CaM03-5\'): 5\'-CCT TCG CAA GAC CCT TCC TCT ATA-3\'

     R-primer (CBH02-3\'): 5\'-GTA GCT GTC GGT GTA GTC CTC GT-3\'

C.  Primer for 2nd detection of CBH351

     F-primer (Cry9C-5\'): 5'-TAC TAC ATC GAC CGC ATC GA-3\'

     R-primer (35Ster-3\'): 5\'-CCT AAT TCC CTT ATC TGG GA- 3\'

(3) Judgement of results

If the PCR amplified reactant of 151 bp is detected for SSIIb-1 primer
to determine the internal standard gene and the PCR amplified reactant
of 170 bp is detected for the primer for 1st detection of CBH351,
conduct PCR reaction of the same DNA extract with use of the primer for
2nd detection of CBH351. If the PCR amplified reactant of 171 bp is
confirmed from this PCR reaction, it is judged as \"CBH351 detected\".
If one of two DNA extract solutions is judged as \"CBH351 detected\",
the relevant sample is finally judged as \"CBH351 detected\". If one of
two DNA extract solutions does not show the PCR amplified reactant
corresponding to the internal standard gene, conduct electrophoresis
again to confirm the PCR amplified reactant. If the PCR amplified
reactant for the internal standard gene is not detected again, ignore
the result obtained from the relevant extraction and use the result from
another DNA extract for judgment. If the PCR amplified reactant for
internal standard gene is not detected in both DNA extracts, conduct DNA
extraction and PCR reaction again. If the PCR amplified reactant for the
internal standard gene is not detected in the re-extracted DNA solution,
it is judged as \"Impossible\". Table 10 shows the example of judgment.

**8) Analysis of GM potatoes (New Leaf Plus, New Leaf Y)**

For GM potatoes, use DNA solution obtained from repeated extraction (2
times) according to the method described in ③ Potatoes and processed
potato products, 3) Extraction and purification of DNA and perform PCR
reaction according to the qualitative PCR method described in 5)
Qualitative analysis method. Use the following primers. Primers for this
test are prepared and sold by Nippon Gene (Refer to Table 2). Other
primers that produce the equivalent results can be used.

(1) Primer for detection of New Leaf Plus

 Use Pss 01n primer to identify the internal standard gene.

    F-primer (Pss 01n-5\'): 5\'-TGA CCT GGA CAC CAC AGT TAT-3\'

    R-primer (Pss 01n-3\'): 5\'-GTG GAT TTC AGG AGT TCT TCG A-3\'

  Primer for 1st detection of New Leaf Plus

    F-primer (p-FMV02-5\'): 5\'-AAA TAA CGT GGA AAA GAG CTG TCC TGA-3\'

    R-primer (PLRV01-3\'): 5\'-AAA AGA GCG GCA TAT GCG GTA AAT CTG-3\'

 Use PLRV-rep1-5\' and PLRV-rep1-3\' as primer for 2nd detection of New
Leaf Plus.

(2) Primer for detection of New Leaf Y

 Use Pss 01n primer to identify the internal standard gene.

    F-primer (Pss 01n-5\'): 5\'-TGA CCT GGA CAC CAC AGT TAT-3\'

    R-primer (Pss 01n-3\'): 5\'-GTG GAT TTC AGG AGT TCT TCG A-3\'

 Use p-FMV05-5\' and PVY02-3\' as primer for 1st detection of New Leaf
Y.

  Use pVY01-5\' and pVY01 -3\' as primer for 2nd detection of New Leaf
Y.

(3) Judgement of results

If the PCR amplified reactant of 216 bp is detected for the primer to
determine the internal standard gene and the PCR amplified reactant of
234 bp and/or 225 bp is detected for the primer for 1st detection of New
Leaf Plus and/or New Leaf Y, conduct PCR reaction of the same DNA
extract with use of the primer for 2nd detection of New Leaf Plus and/or
New Leaf Y. If the PCR amplified reactant of 172 bp and/or 161 bp is
confirmed from this PCR reaction, it is judged as \"New Leaf Plus and/or
New Leaf Y detected\". If one of two DNA extract solutions is judged as
\"New Leaf Plus and/or New Leaf Y detected\", the relevant sample is
finally judged as \"New Leaf Plus and/or New Leaf Y detected\". If one
of two DNA extract solutions does not show the PCR amplified reactant
corresponding to the internal standard gene, conduct electrophoresis
again to confirm the PCR amplified reactant. If the PCR amplified
reactant for the internal standard gene is not detected again, ignore
the result obtained from the relevant extract and use the result from
another DNA extract for judgment. If the PCR amplified reactant for
internal standard gene is not detected in both DNA extracts, conduct DNA
extraction and PCR reaction again. If the PCR amplified reactant for the
internal standard gene is not detected in the re-extracted DNA solution,
it is judged as \"Impossible\". Table 10 shows the example of judgment.

Table 10. Example of judgment: StarLink corn (CBH351) and GM potato

  ----------- ----------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ------
  Items                   1          2          3          4          5          6          7          8          9

  Extract 1   Internal    ＋         ＋         ＋         ＋         ＋         ＋         ＋         ＋         －
              Standard                                                                                            
              Gene                                                                                                

              1st         ＋         ＋         ＋         ＋         －         －         ＋         ＋         /
              detection                                                                                           

              2nd         ＋         ＋         ＋         ＋         /          /          －         －         /
              detection                                                                                           

  Extract 2   Internal    ＋         ＋         ＋         －         ＋         －         ＋         －         －
              Standard                                                                                            
              Gene                                                                                                

              1st         ＋         ＋         －         －         －         －         ＋         －         /
              detection                                                                                           

              2nd         ＋         －         /          /          /          /          －         /          /
              detection                                                                                           

  Judgement               Detected   Detected   Detected   Detected   Not        Not        Not        Not        /
                                                                      Detected   Detected   Detected   Detected   
  ----------- ----------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ------

\*  Sample No. 9 will be subjected to a third extraction.

\*\*  ＋ means \"detected\", － means \"not detected\", and / means
\"test not required\".