Dutta Shanta, Chatterjee A, Dutta P, Rajendran K, Roy S, Pramanik K C, Bhattacharya S K
National Institute of Cholera and Enteric Diseases, Beliaghata, Calcutta 700010 and *University College of Medicine, Calcutta 700024, India.
J Med Microbiol. 2001 Aug;50(8):667-674. doi: 10.1099/0022-1317-50-8-667.
As the sensitivity of the conventional techniques for identifying Shigella spp. and enteroinvasive Escherichia coli (EIEC) causing dysentery cases is low, a PCR assay was evaluated in this study. Analytical sensitivity (2 x 10(2) cfu) of the PCR technique was obtained by artificially spiking negative stool samples with a standard strain of S. flexneri type 2, then determining the detection limit. Specificity (100%) of the method was determined by testing a number of known Shigella and EIEC strains and organisms other than Shigella spp. A total of 300 stool samples collected from children with acute diarrhoea was plated on to two selective agar media after enrichment in Luria broth. Shigella spp. were isolated from 7.7% (23 of 300) and EIEC from 1% (3 of 300) patients. All enriched stool samples were subjected to PCR to amplify the target sequence of invasive plasmid antigen (ipa)H locus, a multicopy element found on the chromosome and invasion plasmid. The stool PCR was positive in 24 of the 26 culture-positive and in 22 culture-negative stools, thus detecting the presence of Shigella spp. or EIEC in 15.3% (46 of 300) of diarrhoea cases. When an ial probe was used for colony hybridistion with enriched stool cultures blotted on to membranes, 9.6% (29 of 300) of dysentery cases were identified as being caused by Shigella spp. or EIEC. Thus the sensitivity of enriched stool culture, colony hybridisation and enriched stool PCR was found to be 54%, 60% and 96%, respectively, when each of the methods was compared to the total microbiologically confirmed cases of dysentery. It was also observed that only 38% (48 of 126) of acute bloody dysentery cases actually had shigella or EIEC infection, as confirmed by laboratory methods. Moreover, this PCR assay could identify a number of untypable Shigella strains (Sh OUT), which would have remained undiagnosed had this assay not been used.
由于用于鉴定引起痢疾病例的志贺氏菌属和侵袭性大肠杆菌（EIEC）的传统技术灵敏度较低，本研究对一种聚合酶链反应（PCR）检测方法进行了评估。通过用2型福氏志贺氏菌标准菌株人工接种阴性粪便样本，然后确定检测限，获得了PCR技术的分析灵敏度（2×10² 菌落形成单位）。通过检测一些已知的志贺氏菌和EIEC菌株以及志贺氏菌属以外的微生物来确定该方法的特异性（100%）。从患有急性腹泻的儿童中收集的300份粪便样本在吕氏肉汤中富集后，接种到两种选择性琼脂培养基上。从7.7%（300例中的23例）患者中分离出志贺氏菌属，从1%（300例中的3例）患者中分离出EIEC。所有富集的粪便样本都进行PCR，以扩增侵袭性质粒抗原（ipa）H基因座的目标序列，该基因座是在染色体和侵袭质粒上发现的多拷贝元件。粪便PCR在26例培养阳性样本中的24例以及22例培养阴性样本中呈阳性，从而在15.3%（300例中的46例）腹泻病例中检测到志贺氏菌属或EIEC的存在。当使用ial探针与点样在膜上的富集粪便培养物进行菌落杂交时，9.6%（300例中的29例）痢疾病例被鉴定为由志贺氏菌属或EIEC引起。因此，当将每种方法与微生物学确诊的痢疾总病例数进行比较时，发现富集粪便培养、菌落杂交和富集粪便PCR的灵敏度分别为54%、60%和96%。还观察到，经实验室方法确认，只有38%（126例中的48例）急性血性痢疾病例实际感染了志贺氏菌或EIEC。此外，这种PCR检测方法可以鉴定出一些无法分型的志贺氏菌菌株（Sh OUT），如果不使用该检测方法，这些菌株将无法诊断。
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