开发一种新的多重 PCR 检测粪便球虫寄生虫。

Development of a new multiplex PCR to detect fecal coccidian parasite.

机构信息

Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, 605 006, India.

Department of Paediatrics, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, 605 006, India.

出版信息

Indian J Gastroenterol. 2023 Apr;42(2):241-248. doi: 10.1007/s12664-022-01315-7. Epub 2023 Mar 17.

DOI:10.1007/s12664-022-01315-7

PMID:36930399

Abstract

BACKGROUND

Cryptosporidium spp., Cystoisospora belli and Cyclospora cayetanensis are common intestinal coccidian parasites causing gastroenteritis. The clinical presentation caused by each parasite is indistinguishable from each other. Uniplex polymerase chain reaction (PCR) for these three groups of intestinal coccidian parasites was developed by us in our laboratory. Thereafter, we planned to develop a single-run multiplex polymerase chain reaction (mPCR) assay to detect Cryptosporidium spp., C. belli and C. cayetanensis simultaneously from a stool sample and described it here as coccidian mPCR.

METHODS

New primers for C. belli and C. cayetanensis were designed and uniplex PCRs were standardized. The coccidian mPCR was standardized with known positive DNA control isolates. It was validated with 58 known positive and 58 known negative stool samples, which were previously identified by uniplex PCR.

RESULTS

The coccidian mPCR was standardized with earlier primers designed by us for Cryptosporidium spp. and C. cayetanensis, and a newly designed primer for the internal transcribed spacer-1 (ITS-1) gene for C. belli. The coccidian mPCR was 92.1% sensitive for Cryptosporidium spp., and 100% sensitive for C. belli and C. cayetanensis each, when tested on 116 known samples. It was 100% specific for all intestinal coccidian parasites. Two representative PCR products of the newly designed ITS-1 primer for C. belli were sequenced and submitted to the GenBank, which best match with the sequences of C. belli.

CONCLUSION

A highly sensitive, specific, cost-effective, indigenous, single-run coccidian mPCR has been developed, which can simultaneously detect Cryptosporidium spp., C. belli and C. cayetanensis.

摘要

背景

隐孢子虫属、贝氏等孢子虫和环孢子虫是常见的引起肠胃炎的肠道球虫寄生虫。每种寄生虫引起的临床表现彼此无法区分。我们实验室开发了一种用于检测这三组肠道球虫寄生虫的单重聚合酶链反应（PCR）。此后，我们计划开发一种单管多重聚合酶链反应（mPCR）检测方法，以同时从粪便样本中检测隐孢子虫属、贝氏等孢子虫和环孢子虫，在此将其描述为球虫 mPCR。

方法

为贝氏等孢子虫和环孢子虫设计了新的引物，并对单重 PCR 进行了标准化。用已知的阳性 DNA 对照分离株对球虫 mPCR 进行了标准化。用 58 个已知阳性和 58 个已知阴性粪便样本进行了验证，这些样本之前已通过单重 PCR 进行了鉴定。

结果

球虫 mPCR 是用我们之前为隐孢子虫属和环孢子虫设计的引物和一个新设计的贝氏等孢子虫内部转录间隔区-1（ITS-1）基因引物进行标准化的。在对 116 个已知样本进行测试时，球虫 mPCR 对隐孢子虫属的灵敏度为 92.1%，对贝氏等孢子虫和环孢子虫的灵敏度均为 100%。它对所有肠道球虫寄生虫均具有 100%的特异性。新设计的用于检测贝氏等孢子虫的 ITS-1 引物的两个代表性 PCR 产物已被测序并提交到 GenBank，与贝氏等孢子虫的序列最佳匹配。