Poon Rosana W S, Tam Emily W T, Lau Susanna K P, Cheng Vincent C C, Yuen Kwok-Yung, Schuster Rolf K, Woo Patrick C Y
Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong.
Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong; State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Pok Fu Lam, Hong Kong; Research Centre of Infection and Immunology, The University of Hong Kong, Pok Fu Lam, Hong Kong; Carol Yu Centre for Infection, The University of Hong Kong, Pok Fu Lam, Hong Kong; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The University of Hong Kong, Pok Fu Lam, Hong Kong.
Diagn Microbiol Infect Dis. 2017 Nov;89(3):185-190. doi: 10.1016/j.diagmicrobio.2017.07.012. Epub 2017 Jul 29.
Unlike bacteria and fungi, identification of helminths by gene sequencing is not well-standardized. No "pan-cestode" or "pan-nematode" PCR primers are available. In this study, we designed 2 pairs of PCR primers for amplifying the cox1 genes of cestodes and nematodes respectively and validated their usefulness for real-time PCR and sequencing identification using clinical samples with cestodes and nematodes collected from a variety of animals and human in 7 countries in Asia, Europe and Africa. The detection limits of the cox1 real-time PCR assays for cestodes and nematodes were 10 copies/reaction of extracted DNA, corresponding to C values of 33 and 31 respectively. Real-time PCR using the 2 pairs of primers and probes showed positive results for all 20 clinical samples of cestodes and nematodes. Using phenotypic identification results as the reference standard, DNA sequencing successfully identified all the 5 cestodes and 7 nematodes with cox1 gene sequences available in GenBank, with all these names appearing as the best match of the cox1 gene sequences of the corresponding clinical samples. The percentage nucleotide identities between the cox1 gene sequences of the samples and those of the corresponding best match sequences in GenBank were 98-100%. For the remaining 5 cestodes and 3 nematodes, the corresponding cox1 gene sequences were not available in GenBank. cox1 gene sequencing is discriminative enough for accurately identifying most of the cestodes and nematodes in the present study. Further expansion of the cox1 gene sequence database will enable accurate identification of more cestodes and nematodes.
与细菌和真菌不同，通过基因测序鉴定蠕虫的方法尚未得到很好的标准化。目前没有“泛绦虫”或“泛线虫”的PCR引物。在本研究中，我们设计了2对PCR引物，分别用于扩增绦虫和线虫的细胞色素c氧化酶亚基1（cox1）基因，并使用从亚洲、欧洲和非洲7个国家的各种动物和人类中收集的绦虫和线虫临床样本，验证了它们在实时PCR和测序鉴定中的有效性。绦虫和线虫的cox1实时PCR检测限为提取DNA的10拷贝/反应，对应的C值分别为33和31。使用这2对引物和探针进行的实时PCR对所有20份绦虫和线虫临床样本均显示阳性结果。以表型鉴定结果作为参考标准，DNA测序成功鉴定了GenBank中具有cox1基因序列的所有5种绦虫和7种线虫，所有这些名称均显示为相应临床样本cox1基因序列的最佳匹配。样本的cox1基因序列与GenBank中相应最佳匹配序列之间的核苷酸同一性百分比为98-100%。对于其余5种绦虫和3种线虫，GenBank中没有相应的cox1基因序列。在本研究中，cox1基因测序具有足够的鉴别力，能够准确鉴定大多数绦虫和线虫。进一步扩大cox1基因序列数据库将能够准确鉴定更多的绦虫和线虫。
