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目的建立沙门菌PCR-ELISA检测方法,并进行验证。方法根据沙门菌inv A基因设计PCR-ELISA引物及探针,进行PCR扩增、核酸杂交及ELISA检测,对变性反应条件、杂交探针浓度、杂交时间和温度及显色时间进行优化,确定阴性、阳性判定的阈值,并进行敏感性、特异性及重复性验证。用建立的PCR-ELISA方法检测人工染菌的22批中药丸剂及27批液体乳中的沙门菌,并与PCR及GB4789方法进行比较。结果 150 mmol/L Na OH变性液可使核酸变性最彻底,50 pmol/ml地高辛标记探针为最适浓度,50℃60 min为最佳杂交时间和温度,最佳显色时间为50 min;阴性、阳性的阈值为0.265。该方法最低检测限为0.5 pg/μl,灵敏度高;能针对性地扩增沙门菌的特异性片段,检测特异性强;检测结果的批内、批间变异系数均小于10%,重复性较好。人工染菌的22批中药丸剂和27批液体乳的PCR-ELISA方法检测阳性率明显高于PCR法,与GB4789方法检测阳性率基本一致。结论优化后的PCRELISA方法特异性强,灵敏度高,重复性好,可快速检测食品、药品中的沙门菌。
Objective To establish a PCR-ELISA method for the detection of Salmonella and validate it. Methods PCR-ELISA primers and probes were designed according to inv A gene of Salmonella. PCR amplification, nucleic acid hybridization and ELISA were used to detect the denaturation reaction conditions, hybridization probe concentration, hybridization time and temperature, and colorimetric time. Negative , Positive judgment threshold, and sensitivity, specificity and repeatability verification. Twenty-two batches of Chinese medicine pellets and twenty-seven batches of Salmonella in liquid milk were detected by PCR-ELISA method and compared with PCR and GB4789 method. Results 150 mmol / L Na OH denatured solution could denature the nucleic acid most thoroughly. The optimal concentration of 50 pmol / ml digoxigenin-labeled probe was 50 min at 60 min for optimal hybridization time and temperature. The optimal color development time was 50 min; negative, positive threshold of 0.265. The method has the lowest detection limit of 0.5 pg / μl and high sensitivity. It can specifically amplify the specific fragments of Salmonella and has strong specificity. The intra-assay and inter-assay CVs of the assay are less than 10% it is good. The positive rate of PCR-ELISA of 22 batches of Chinese medicine pellets and 27 batches of liquid emulsion was significantly higher than that of PCR method, which was consistent with the detection rate of GB4789. Conclusion The optimized PCRELISA method has high specificity, high sensitivity and good repeatability, and can rapidly detect Salmonella in food and medicine.