Nastasi A, Mammina C, Mioni R
Department of Hygiene and Microbiology G. D'Alessandro, Università degli Studi, Palermo, Italy.
New Microbiol. 1999 Jul;22(3):195-202.
A rapid and sensitive PCR-hybridization procedure for detection of Salmonella serovars in food samples was developed. This method is based on three subsequent steps: (1) extraction of nucleic acids from a 2 ml aliquot of the pre-enrichment medium used for the conventional culture method after 6 h of incubation at 37 degrees C; (2) amplification with primers selected from the sequences of invE and invA genes; (3) Southern blot and hybridization with a biotin labeled oligonucleotide probe. The entire procedure requires 30 h. The PCR-hybridization assay was able to detect as little as 50 fg of purified chromosomal DNA of S. typhimurium and 0.2 cfu g-1 of an artificially contaminated food sample. Of 245 food samples analyzed by culture and PCR-hybridization, 20 were positive by both methods and 16 were positive by PCR-hybridization only. None of the 209 PCR-negative samples tested positive by culture. The sensitivity, specificity, alpha and beta error values of the results of the PCR-hybridization procedure, compared with those of culture, were 100, 92.9, 0 and 7.1%, respectively. These results indicate that a short pre-enrichment and PCR-hybridization could be used as a screening test for the detection of Salmonella in food samples.
开发了一种用于检测食品样本中沙门氏菌血清型的快速灵敏的聚合酶链反应-杂交方法。该方法基于三个后续步骤：（1）在37℃孵育6小时后，从用于传统培养方法的2毫升预富集培养基等分试样中提取核酸；（2）用从invE和invA基因序列中选择的引物进行扩增；（3）Southern印迹法并用生物素标记的寡核苷酸探针进行杂交。整个过程需要30小时。聚合酶链反应-杂交测定法能够检测到低至50 fg的鼠伤寒沙门氏菌纯化染色体DNA和0.2 cfu g-1的人工污染食品样本。在通过培养和聚合酶链反应-杂交分析的245个食品样本中，两种方法均为阳性的有20个，仅聚合酶链反应-杂交为阳性的有16个。209个聚合酶链反应阴性样本经培养均未呈阳性。与培养结果相比，聚合酶链反应-杂交程序结果的灵敏度、特异性、α和β误差值分别为100%、92.9%、0和7.1%。这些结果表明，短时间预富集和聚合酶链反应-杂交可作为食品样本中沙门氏菌检测的筛选试验。
