The study results on previous and new extraction methods
for Loopamp® Salmonella Detection Kit
Since the release of Loopamp® Salmonella Detection Kit in March 2003, it has been adopted for food inspection by
many testing centers, food manufacturers etc. However, samples with high lipid content such as poultry orcompressed-oil residuals, might inhibit LAMP reactions and show negative results even though culture methods show
positive results.
Eiken Chemical Co., Ltd. has been researching on improving the extraction method. We here would like to introduce asolution that not only better solve the inhibition problem on high lipid samples, but also shorten the reaction time for thedetectable samples by previous extraction method (hereinafter referred to as previous method), and show a more
prominent turbidity increase of magnesium pyrophosphate precipitate. We adopt this method as the new extraction
method (hereinafter referred to as improved method). Previous method was known to have interference from EEM
Bouillon Media, but this interference has been avoided in improved method.
As a result, improved method has been adopted for handling all the samples, and the contents of the kit, operation
procedures and package insert has been modified accordingly to reflect the changes. The study results on the previousand new extraction methods using different samples and enrichment media are summarized as follows.
1. The operation procedures of improved method
Apart from the procedures of previous method, improved method contains the procedures such as centrifuge andremoving supernatant, neutralization after heat treatment etc.
First, dispense 50µL of the pre-enrichment culture into each tube as samples, and centrifuge for 5 minutes. Thenremove the supernatant carefully as to not convolute the precipitate. If there is no precipitate in the solution, remove
40µL of supernatant. Add 80µL of Extraction Solution for Foods (EX F), well mix and spin down for a few seconds,
and then incubate at 95°C for 5 minutes. Add 10µL of 1M Tris-HCl: pH7.0 (Tris) and well mix, centrifuge for 30
seconds at room temperature. The supernatant will be used as sample solution (Figure below. Refer to the package
insert for more details).

Previous method
Pre-enrichment

Improved method
Pre-enrichment
Food 25g + BPW or EEM Bouillon Media
225mL (36 ± 1°C, 20-24 hours)

Food 25g + BPW225mL（36 ± 1°C, 20-24 hours）*: For the pre-enrichment of liver related materials, EEM

Mix 50µL of EX F and 50µL of pre-enrichment
culture, spin down.

Centrifuge 50µL of pre-enrichment culture for 5minutes and remove the supernatantBouillon Media should be used.

Add EX F 80µL, well mix and spin down
Heat treatment (95°C for 5 minutes)

Heat treatment (95°C for 5 minutes)
Add 10µL of Tris and well mix.

Centrifuge (at room temperature for 1minute)Centrifuge (at room temperature for 30 seconds)

The supernatant will be used as sample solution

The supernatant will be used as sample solution

2. Study on extraction methods[Method of study]Mix 25g of sample with 225g of liquid enrichment media for Salmonella (Buffered Peptone Water (hereinafterreferred to as BPW) or EEM Bouillon Media (hereinafter referred to as EEM)). After stomachering, incubate at 37°C for
24 hours (in some cases 16 hours) for enrichment. And then prepare the stimulated samples to contain 2.4 X 105
CFU/mL or 2.4 X 104 CFU/mL of Salmonella Enteritidis.
Use previous method and improved method respectively to conduct the extraction from the simulated samples, and take5µL (600 CFU/test or 60 CFU/test) each for the LAMP reactions.
The results of LAMP reactions using different food materials and enrichment media are summarized as follows.
1) The study on poultry samples
With the previous method, lipid content from poultry might interfere LAMP reactions. Samples were cultured by BPWMedia in this study, although previous method showed inhibition in turbidity increase, improved method showed a clearturbidity increase pattern.
<Previous method><Improved method>

2.4X105 CFU/mL (600 CFU/test)
2.4X104 CFU/mL ( 60 CFU/test)
0 CFU/mL ( 0 CFU/test)

2) The study on pork, beef and beef liver samples
When meat samples were cultured by BPW Media, although previous method showed interference in both pork andbeef samples, improved method showed a clear turbidity pattern and the amplification starting time could also beobserved earlier. When cultured by EEM Media, previous method has shown more severe interference than its BPWresults. In contrast, when improved method was used, although the starting time of amplification is slower than the ones
using BPW Media, its amplification was recognized much faster than the previous method and the amplification could beclearly confirmed.
<Previous method><Improved method>

2.4X105 CFU/mL (600 CFU/test)
2.4X104 CFU/mL ( 60 CFU/test)
0 CFU/mL ( 0 CFU/test)

In the study of beef liver, pre-enrichment were conducted with 4 samples (sample A, B, C, D) for 16 hours, and thenmixed with the bacteria solution (2.4 X105CFU/mL). When BPW Media were used, inhibition to reaction was observedin previous method, and in some cases, amplification could not be recognized within 60 minutes. When BPW Media
were used with improved method, some samples even showed more severe inhibition. However, when EEM was usedwith improved method, although previous method showed more significant inhibition in EEM than BPW Media,
improved method did not have any problem in all the detections. Therefore, EEM Media should be used for thepre-enrichment of beef liver samples.
<Previous method><Improved method>Beef Liver A
Beef Liver B
Beef Liver C
Beef Liver D3) The study on egg samples
Whole egg and yolk only were cultured in BPW Media. Although amplification could be observed in previous method,
turbidity increase was significantly inhibited. When improved method was used, not only turbidity increase became more
prominent, the amplification starting time could also be observed earlier. It seems that the inhibition elements to LAMP
reactions in the enrichment culture had been removed by centrifuge operation.
<Previous method>
BPW + Yolk

<Improved method>
BPW + Yolk

2.4X105 CFU/mL (600 CFU/test)
2.4X104 CFU/mL ( 60 CFU/test)4) The study on using only enrichment media (BPW or EEM)
Incubated only the enrichment media at 37°C for 24 hours and added bacteria into them. For BPW Media withimproved method, the amplification starting time could be observed earlier than previous method. When EEM Mediawas used, although previous method did not show amplification at 60CFU/test within 60 minutes, improved method
showed the turbidity increase after about 25 minutes of the amplification.
<Improved method><Improved method>

2.4X105 CFU/mL (600 CFU/test)
2.4X104 CFU/mL ( 60 CFU/test)5) The study on compressed-oil residual samples
In the study of compressed-oil residuals, cultured bacteria were added into the BPW Media. For the compressed-oil
residuals of wheat germ, while previous method showed some inhibition, improved method showed better turbidity
increase. Improved method also showed more prominent amplification curves and the amplification starting time could
be observed earlier. For the compressed-oil residuals of coleseed, although previous method did not show any turbidity
increase, improved method showed the turbidity increase after about 20 minutes of the amplification.
<Previous method>Compressed-oilresiduals(Wheatgerm)

Compressed-oilresiduals(coleseed)<Improved method>Compressed-oilresiduals(Wheatgerm)

Compressed-oilresiduals(coleseed)

2.4X105 CFU/mL (600 CFU/test)
2.4X104 CFU/mL ( 60 CFU/test)