Lin Chien-Ku, Hung Cho-Lien, Chiang Yu-Cheng, Lin Chi-Ming, Tsen Hau-Yang
Department of Food Science and Nutrition, Hung-Kuang University, Shalu, Taichung County, Taiwan 433, China.
Int J Food Microbiol. 2004 Nov 1;96(2):205-14. doi: 10.1016/j.ijfoodmicro.2004.03.027.
Previously, we have reported a 16S rDNA targeted polymerase chain reaction (PCR) method for the specific detection of Salmonella serovars [J. Appl. Bacteriol. 80 (1996) 659]. The target sites of its primers, i.e. 16SFI and 16SIII, according to the data in GenBank, were found mismatched to the corresponding sequences of some Salmonella serovars, such as those of S. Houten, S. Chingola, S. Bareilly, and S. Weltevreden. Accordingly, a PCR method using a nonspecific primer MINf combined with a primer modified from our 16SFI primer, i.e. the primer MINr, was developed and displayed better detection specificity [Int. J. Food Microbiol. 80 (2003) 67]. In this study, we show the sequence heterogenicity at the primer 16SFI targeting sites for some Salmonella serovars. Thus, the sequence used for designing of PCR primers might be just one of the several possible sequences. Such a situation may lead to the misjudgment on evaluation of the specificity of the primers if this was only based on the data in GenBank. Strains of the above described Salmonella serovars with target sequences from GenBank mismatched to the primer 16SF1 were reidentified and their PCR results were confirmed. Meanwhile, their 16SFI/16SIII primer annealing sites were sequenced and the sequences obtained were found completely and highly homologous to those of 16SFI and complementary to those of 16SIII primer, respectively.
此前，我们报道了一种用于特异性检测沙门氏菌血清型的16S rDNA靶向聚合酶链反应（PCR）方法[《应用细菌学杂志》80 (1996) 659]。根据GenBank中的数据，其引物16SFI和16SIII的靶位点被发现与一些沙门氏菌血清型的相应序列不匹配，例如霍滕沙门氏菌、钦戈拉沙门氏菌、巴雷利沙门氏菌和韦尔泰夫reden沙门氏菌的序列。因此，开发了一种使用非特异性引物MINf与从我们的16SFI引物修饰而来的引物（即引物MINr）相结合的PCR方法，该方法显示出更好的检测特异性[《国际食品微生物学杂志》80 (2003) 67]。在本研究中，我们展示了一些沙门氏菌血清型在引物16SFI靶向位点的序列异质性。因此，用于设计PCR引物的序列可能只是几种可能序列之一。如果仅基于GenBank中的数据，这种情况可能会导致对引物特异性评估的错误判断。对上述GenBank靶序列与引物16SF1不匹配的沙门氏菌血清型菌株进行了重新鉴定，并确认了它们的PCR结果。同时，对它们的16SFI/16SIII引物退火位点进行了测序，发现获得的序列分别与16SFI的序列完全且高度同源，与16SIII引物的序列互补。
