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首页> 外文期刊>Applied and Environmental Microbiology >Direct Quantification of Campylobacter jejuni and Campylobacter lanienae in Feces of Cattle by Real-Time Quantitative PCR
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Direct Quantification of Campylobacter jejuni and Campylobacter lanienae in Feces of Cattle by Real-Time Quantitative PCR

机译：实时定量PCR直接定量牛粪中的空肠弯曲菌和兰氏弯曲杆菌

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Campylobacter species are fastidious to culture, and the ability to directly quantify biomass in microbiologically complex substrates using real-time quantitative (RTQ) PCR may enhance our understanding of their biology and facilitate the development of efficacious mitigation strategies. This study reports the use of nested RTQ-PCR to directly quantify Campylobacter jejuni and Campylobacter lanienae in cattle feces. For C. jejuni, the single-copy mapA gene was selected. For C. lanienae, the three-copy 16S rRNA gene was targeted. RTQ-PCR primers were tested alone or they were nested with species-specific primers, and amplification products were detected using the intercalating dye SYBR Green. Nesting did not increase the specificity or sensitivity of C. jejuni quantification, and the limit of quantification was 19 to 25 genome copies (≈3 × 103 CFU/g of feces). In contrast, nested RTQ-PCR was necessary to confer specificity on C. lanienae by targeting the 16S rRNA gene. The limit of quantification was 1.8 genome copies (≈250 CFU/g of feces), and there was no discernible difference between the two C. lanienae secondary primer sets evaluated. Detection and quantification of C. jejuni in naturally infested cattle feces by RTQ-PCR were comparable to the results of culture-based methods. In contrast, culturing did not detect C. lanienae in 6 of 10 fecal samples positive for the bacterium and substantially underestimated cell densities relative to nested RTQ-PCR. The results of this study illustrate that RTQ-PCR can be used to directly quantify campylobacters, including very fastidious species, in a microbiologically and chemically complex substrate. Furthermore, targeting of a multicopy universal gene provided highly sensitive quantification of C. lanienae, but nested RTQ-PCR was necessary to confer specificity. This method will facilitate subsequent studies to elucidate the impact of this group of bacteria within the gastrointestinal tracts of livestock and studies of the factors that influence colonization success and shedding.
机译：弯曲杆菌属菌种易于培养，使用实时定量（RTQ）PCR直接定量微生物复杂底物中生物量的能力可能会增强我们对其生物学的了解，并促进有效缓解策略的发展。这项研究报告了使用嵌套式RTQ-PCR直接定量牛粪中的空肠弯曲杆菌和拉曼弯曲杆菌。对于空肠弯曲菌，选择单拷贝mapA基因。对于兰念珠菌，靶向三拷贝16S rRNA基因。单独测试RTQ-PCR引物，或将其与种特异性引物嵌套，并使用嵌入染料SYBR Green检测扩增产物。筑巢并没有增加空肠弯曲杆菌定量的特异性或敏感性，并且定量限为19至25个基因组拷贝（≈3×103 CFU / g粪便）。相反，通过靶向16S rRNA基因，嵌套式RTQ-PCR才能赋予兰科念珠菌特异性。定量限为1.8个基因组拷贝（≈250CFU / g粪便），并且所评估的两个兰氏梭菌二级引物组之间没有明显的差异。通过RTQ-PCR对自然感染的牛粪中空肠弯曲杆菌的检测和定量与基于培养的方法的结果相当。相反，在嵌套培养的10份粪便样本中有6份对细菌呈阳性，相对于巢式RTQ-PCR而言，细胞密度大大低估了，培养物中未检测到兰氏梭菌。这项研究的结果表明，RTQ-PCR可用于在微生物和化学复杂的底物中直接定量弯曲杆菌，包括极富挑战性的物种。此外，对多拷贝通用基因的靶向提供了兰氏梭菌的高度灵敏定量，但是巢状RTQ-PCR对于赋予特异性是必需的。这种方法将有助于随后的研究，以阐明这组细菌在牲畜胃肠道中的影响以及对影响定殖成功和脱落的因素的研究。

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