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首页> 外文期刊>Journal of Clinical Microbiology >Pentaplex PCR Assay for Detection of Hemorrhagic Bacteria from Stool Samples
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Pentaplex PCR Assay for Detection of Hemorrhagic Bacteria from Stool Samples

机译：五倍体PCR检测粪便中出血性细菌的检测

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Diarrheal diseases cause illness and death among children younger than 10 years in developing countries. Conventional testing for the detection of hemorrhagic bacteria takes 2 to 5 days to yield complete information on the organism and its antibiotic sensitivity pattern. Hence, in the present study, we developed a molecular-based diagnostic assay that identifies common hemorrhagic bacteria in stool samples. A set of specific primers were designed for the detection of Salmonella spp., Shigella spp., enterohemorrhagic Escherichia coli (EHEC), and Campylobacter spp., suitable for use in a one-tube PCR assay. The assay in the present study simultaneously detected five genes, namely, ompC for the Salmonella genus, virA for the Shigella genus, eaeA for EHEC, 16S rRNA for the Campylobacter genus, and hemA for an internal control. Specific primer pairs were successfully designed and simultaneously amplified the targeted genes. Validation with 20 Gram-negative and 17 Gram-positive strains yielded 100% specificity. The limit of detection of the multiplex PCR assay was 1 × 103 CFU at the bacterial cell level and 100 pg at the genomic DNA level. Further evaluation of the multiplex PCR with 223 bacterium-spiked stool specimens revealed 100% sensitivity and specificity. We conclude that the developed multiplex PCR assay was rapid, giving results within 4 h, which is essential for the identification of hemorrhagic bacteria, and it might be useful as an additional diagnostic tool whenever time is important in the diagnosis of hemorrhagic bacteria that cause diarrhea. In addition, the presence of an internal control in the multiplex PCR assay is important for excluding false-negative cases.
机译：腹泻病在发展中国家引起10岁以下儿童的疾病和死亡。用于检测出血性细菌的常规测试需要2到5天才能得出有关该生物及其抗生素敏感性模式的完整信息。因此，在本研究中，我们开发了一种基于分子的诊断测定法，可鉴定粪便样本中的常见出血细菌。设计了一组特异性引物，用于检测沙门氏菌，志贺氏菌，肠出血性大肠杆菌（EHEC）和弯曲杆菌属，适用于单管PCR分析。本研究同时检测了五个基因，分别是沙门氏菌属的 ompC ，志贺氏菌属的 virA ，EHEC的 eaeA 。弯曲杆菌属的16S rRNA，内部对照的 hemA 。成功设计了特异性引物对，并同时扩增了目标基因。用20株革兰氏阴性和17株革兰氏阳性菌株进行验证可产生100％的特异性。多重PCR检测的检出限在细菌细胞水平为1×10 3 CFU，在基因组DNA水平为100 pg。用223种细菌标本粪便标本进行的多重PCR的进一步评估显示出100％的敏感性和特异性。我们得出的结论是，开发的多重PCR检测快速，可在4小时内给出结果，这对于鉴定出血性细菌至关重要，并且只要时间在导致腹泻的出血性细菌的诊断中很重要，它就可以用作其他诊断工具。另外，多重PCR分析中内部对照的存在对于排除假阴性病例很重要。

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