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首页> 外文期刊>Journal of Clinical Microbiology >An enzyme-linked immunosorbent assay to detect PCR products of the rfbS gene from serogroup D salmonellae: a rapid screening prototype.
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An enzyme-linked immunosorbent assay to detect PCR products of the rfbS gene from serogroup D salmonellae: a rapid screening prototype.

机译：一种酶联免疫吸附测定，可检测D群沙门氏菌rfbS基因的PCR产物：快速筛选原型。

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We describe a digoxigenin-based enzyme-linked immunosorbent assay (DIG-ELISA) following a PCR to detect the amplified lipopolysaccharide rfbS gene as a means for rapid screening of serogroup D salmonellae in stool specimens. For pure bacterial cultures, the sensitivity of the PCR DIG-ELISA was approximately 10 bacteria. In the presence of stool materials, the salmonellae were first isolated by an immunomagnetic separation technique with an O9-specific monoclonal antibody. MATy-O9, followed by PCR and DIG-ELISA. The corresponding sensitivity was about 10 to 100 bacteria. To evaluate the assay performance clinically, 203 stool samples from patients with diarrhea were subjected to the routine culture techniques and the PCR ELISA method with overnight enrichment. The conventional culture method identified 145 salmonellae (31 serogroup B, 27 serogroup C, 83 serogroup D, and 5 serogroup E isolates) and 58 non-salmonella bacteria. The PCR ELISA method correctly identified all 82 serogroup D salmonellae (A405 by ELISA, 2.54 +/- 0.74) but was negative for the other Salmonella serogroups (A405, 0.26 +/- 0.08; n = 63) and non-Salmonella isolates (A405, 0.16 +/- 0.04; n = 58). In order to obtain a visible result, the assay takes approximately 6 h (PCR, 4 h; ELISA, 2 h), along with brief enrichment cultivation of the samples (from 4 to 16 h). Thus, the PCR DIG-ELISA offers a fast, accurate, semiquantitative means of detecting infectious agents such as salmonellae, and future robotic automation is possible.
机译：我们描述了一种基于地高辛配基的酶联免疫吸附测定（DIG-ELISA），经过PCR检测扩增的脂多糖rfbS基因，作为快速筛选粪便样本中血清D组沙门氏菌的手段。对于纯细菌培养，PCR DIG-ELISA的灵敏度约为10个细菌。在粪便存在的情况下，沙门氏菌首先通过免疫磁分离技术与O9特异性单克隆抗体分离。 MATy-O9，然后进行PCR和DIG-ELISA。相应的敏感度约为10至100个细菌。为了临床评估测定性能，对203例腹泻患者的粪便样本进行了常规培养技术和过夜富集的PCR ELISA方法。常规培养方法鉴定出145株沙门氏菌（31个血清群B，27个血清群C，83个D血清群和5个E血清群）和58个非沙门氏菌。 PCR ELISA方法正确鉴定了所有82个D血清型沙门氏菌（通过ELISA测得的A405,2.54 +/- 0.74），但对其他沙门氏菌血清群（A405，0.26 +/- 0.08; n = 63）和非沙门氏菌分离株（A405）阴性，0.16 +/- 0.04; n = 58）。为了获得可见的结果，测定需要大约6小时（PCR，4小时; ELISA，2小时），同时进行简短的样品富集培养（4至16小时）。因此，PCR DIG-ELISA提供了一种快速，准确，半定量的方法来检测诸如沙门氏菌的传染原，并且未来的机器人自动化是可能的。

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