United States Department of Agriculture

Agricultural Marketing Service

Federal Grain Inspection Service

Criteria & Specifications May 17, 2017

Distribution: Industry Originating Office: Technology and Science Division

Biotechnology Test Kit Criteria Supporting Document

Contents

COVER PAGE �������������������������������������������������������������������������������������������������������������������������1

1. BACKGROUND �������������������������������������������������������������������������������������������������������������������

2. ISSUE ����������������������������������������������������������������������������������������������������������������������������������

3. CONCLUSIONS �������������������������������������������������������������������������������������������������������������������

The U.S. Department of Agriculture (USDA) prohibits discrimination in its programs on the basis of race, color, 

national origin, sex, religion, age, disability, political beliefs, and marital or familial status. (Not all prohibited 

bases apply to all programs.) Persons with disabilities who require alternate means for communication of program 

information (Braille, large print, audiotape, etc.) should contact USDA’s TARGET Center at (202) 720-2600 (voice and 

TDD).

To file a complaint, write to the USDA, Office of Civil Rights, Room 326-W, 1400 Independence Avenue, SW, 

Washington, DC 20250-9410, or call (202) 720-5964 (voice and TDD). USDA is an equal employment opportunity 

employer.
Biotechnology Test Kit Criteria 

Supporting Document 

Effective May 17, 2017

Page 2

1. BACKGROUND 

To meet the grain industry’s demand for accurate and reliable qualitative testing for 

genetically engineered (GE) traits in grains and oilseeds, test kit manufacturers have 

asked the United States Department of Agriculture’s Federal Grain Inspection Service 

(FGIS) to maintain a program to verify test kit performance claims and subsequently 

issue Certificates of Performance (COP) for test kits meeting these claims. In response 

to this request, FGIS has established a program for verifying the performance of these 

qualitative rapid test kits

2. ISSUE 

Test kit manufacturers have approached FGIS with their concern that sourcing 100% 

conventional grain to serve as control material for sample analysis is becoming 

increasingly difficult. FGIS understands these concerns given the increasing prevalence 

of adventitious presence of GE content in grain. Therefore, FGIS has applied a

statistical approach to modify the manufacturer’s data submission requirements

A component of the test kit verification process requires the manufacturer to submit test 

data to FGIS. Relative to previous requirements, FGIS has increased the number of

independent tests to 156 and now allows up to 3 failures for both control and fortified 

samples. At this failure rate, the test kit will be verified to have a 95% sensitivity and 

specificity at 95% confidence level. 

• Control Samples

One hundred fifty-six (156) independent analyses performed using thirteen 

(13) different samples tested across three (3) manufacturing lots, at four (4) 

replicates per sample. Up to three (3) false positive results for the genetically

engineered material of interest are allowed

• Fortified Samples

One hundred fifty-six (156) independent analyses performed using thirteen 

(13) different samples fortified at the claimed detection threshold. Samples 

will be tested across three (3) manufacturing lots, at four (4) replicates per 

sample. Up to three (3) false negative results for the genetically engineered

material of interest are allowed
Biotechnology Test Kit Criteria 

Supporting Document 

Effective May 17, 2017

Page 3

3. CONCLUSIONS

a. GE-Free Base Grain Sample

FGIS asserts that the kit manufacturer should make every effort to provide 

GE-free grain samples for the purpose of test kit verification. If GE-free base 

material cannot be sourced, the concentration of GE- grain should be one 

order of magnitude smaller than the LOD of the protein-based kit, or two orders 

of magnitude smaller than the LOD of the PCR-based kit, under the critical 

assumption of sampling m grains and the LOD of PCR is 1/m

(1) For protein-based kits, which are generally one order of magnitude less 

sensitive than PCR-based kits, if the kit can detect 1 GE grain out 1,000 

grains, the base grain samples must contain 1 or fewer GE-grains out 

of 10,000 grains. Assuming the sample is 10,000 grains, this level is not

expected to adversely affect the verification of the 156 control and fortified 

samples. Table 1 shows that if the concentration of GE-grain in the base 

sample is one order of magnitude smaller than the LOD of the kit, the 

probability of detecting a positive sample due to adventitious presence is 

negligible (Table 1)

Table 1. Probabilities of detecting a positive sample by the test kit when the concentration of 

GE- grain is 1 ⁄ m and the LOD of the kit is 10 ⁄ m , assuming m grains sampled for test

Grains in Sample (m) 100 500 1,000 5,000 10,000 50,000

Probability of “+” 

Sample 7.6 × 10−8 1.0 × 10−7 1.1 × 10−7 1.1 × 10−7 1.1 × 10−7 1.1 

*Based on a binomial distribution. Similar results can also be obtained by Poisson distributio

(2) For PCR-based kits, if and sample size is m and the LOD of the kit is 1/m, no 

matter how big is m, the contamination level should be two orders of 

magnitudes smaller than the LOD, that is, < 1 / (100×m), to obtain reasonably 

small number of false positives from the 156 controls (Table 2).

Table 2. Probabilities of detecting a positive sample by the test kit when the concentration 

of GE- grain is one or more orders of magnitude smaller than the LOD of the kit (1 ⁄ m), 

assuming m grains sampled for test

Grains in Sample (m) 100 500 1,000 5,000 10,000 50,000

cont. 1 ⁄ 10m 0.0952 0.0952 0.0952 0.0952 0.0952 0.095

1 ⁄ 100m 0.00995 0.00995 0.00995 0.00995 0.00995 0.00995

1 ⁄ 1000m 9.9 × 10−4 9.9 × 10−4 9.9 × 10−4 9.9 × 10−4 9.9 × 10−4 9.9 × 10−4

1 ⁄ 10000m 9.9 × 10−5 9.9 × 10−5 9.9 × 10−5 9.9 × 10−5 9.9 × 10−5 9.9 × 10−5

*Based on a binomial distribution. Similar results can also be obtained by Poisson distributio
Biotechnology Test Kit Criteria 

Supporting Document 

Effective May 17, 2017

Page 4

Compare Table 1 and Table 2, it is not difficult to see that the size of grain sample 

(m), not the working mechanism of the kit—whether it is based on protein or 

DNA—is the critical factor determining the probability of false positives

• If the LOD of the kit is 1⁄x or 1 out of x grains, the number of grains sampled 

for test should be m=10×x. With this way to determine m, the adventitious 

presence of GE grains in the base sample at the level less than1⁄m will 

contribute negligibly the occurrence of false positive result

• If the LOD of the kit is 1⁄x, and the number of grains sampled for test is m=x, 

the probability of adventitious presence of GE grains in the base sample 

increases dramatically. This probability reaches about 1⁄k if the level of GE 

contamination is 1⁄((k×m)). The probability of false positive is no longer 

negligible if the contamination level is about one order of magnitude smaller 

than the LOD

In either strategy to determine m, there is no way to prepare negative control 

samples if the concentration of GE-grain in the base sample is close to LOD of the 

test kit. The manufacturer is advised to find an alternative source of material.

b. Sensitivity and Specificity of Test

The following calculation provides an estimate to determine the sample sizes 

required for calculating sensitivity and specificity. At a 95% confidence level, the 

minimal sensitivity varies along the number of negative samples among the n 

(=156) fortified samples tested by the kit. If we want a minimal sensitivity of 0.95, 

then the number of false negative among 156 fortified samples must be no more 

than 3 (Table 2)

Table 3. Relationship between observed false negatives and sensitivity

# Negative samples Sensitivity

0 0.981

1 0.970

2 0.960

3 0.951

4 0.942

5 0.934

The number of false positive samples allowed for a given level of specificity can be 

found from the table below. At a 95% confidence level, the minimal specificity varies 

along the number of positive samples among the n (=156) base control samples tested 

by the kit. If we want a minimal specificity of 0.95, then the number of false positives 

among 156 base control samples must be no more than 3 (Table 4)
Biotechnology Test Kit Criteria 

Supporting Document 

Effective May 17, 2017

Page 5

Table 4. Relationship between observed false positives and specificity

# Negative samples Sensitivity

0 0.981

1 0.970

2 0.960

3 0.951

4 0.942

5 0.934