Development of Multiplex PCR Method Utilizing Pooled Sample for Screening of Virulent Enterobacteria

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概要

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• For screening Salmonella, enterohemorrhagic E. coli (EHEC) and Shigella positive samples from a lot of fecal samples, a method using pooled samples was examined. We chose multiplex PCR for the detection of these pathogenic enterobacteria, which does not require DNA extraction and purification, or pre-amplification by culture.1) By using the multiplex PCR, one Salmonella-positive sample containing 7×104cfu/g could be detected from 100-pooled samples. Likewise, one EHEC-positive sample containing 7×104cfu/g and one Shigella-positive containing 4×104cfu/g could be detected from 100-pooled samples. Since the limit of detection in the culture method was 7×104cfu/g for Salmonella and EHEC and 4×105cfu/g for Shigella, they can be detected at the equal sensitivity by the multiplex PCR from a pooled sample less than 100.2) In order to verify the possibility that dead bacteria are detected by the multiplex PCR, a model fecal sample in which genomic DNA was spiked was prepared. As a result, the genomic DNA spiked to the feces was not detected by the multiplex PCR. It was therefore considered less possible that the dead bacteria, of which genomic DNA was eluted into the feces, may be detected.3) From 2,000 of fecal samples, 40 pools containing 50 samples each were prepared. These pools were subjected to the multiplex PCR for screening. Each sample in the pool evaluated to be positive was subjected to the culture method. On the other hand, each of the 2,000 samples was subjected to the culture method without screening. As concern the correlation between with and without the screening, a positive agreement ratio and negative agreement ratio were 100% and 99.95%, respectively.These results indicate that the method examined in this study was useful for screening positive samples from a lot of samples for fecal test.

• 日本食品微生物学会の論文

著者

• 加藤 文男

  東邦大学薬学部

• 井上 浩明

  東洋紡績株式会社

• 萩原 直樹

  株式会社らいふ

• 荒川 琢

  東洋紡績株式会社

• 西井 重明

  東洋紡績株式会社

• 東隆 寛

  東洋紡績株式会社

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• プールした糞便検体を使用したマルチプレックスPCR法によるサルモネラ, 腸管出血性大腸菌, 赤痢菌のスクリーニング法の開発
• Development of Multiplex PCR Method Utilizing Pooled Sample for Screening of Virulent Enterobacteria

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