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核心技术专利：CN118964589B侵权必究
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一种用于同时鉴定和定量马提尼克利什曼原虫与东方利什曼原虫/尚氏利什曼原虫，并检测和定量临床样本中锥虫的一步多重定量聚合酶链反应检测法。

A one-step multiplex qPCR assay for simultaneous identification and quantification of Leishmania martiniquensis and Leishmania orientalis/Leishmania chancei and detection and quantification of trypanosomatids in clinical samples.

作者信息

Mano Chonlada, Hirunpatrawong Piyapha, Prasertsilp Patiya, Kaewmee Saowalak, Limprasert Parinda, Siriyasatien Padet, Tantiworawit Adisak, Gatherer Derek, Urbaniak Michael D, Bates Paul A, Jariyapan Narissara

机构信息

Center of Excellence in Vector Biology and Vector-Borne Disease, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.

Department of Medical and Public Health Secretary, College of Allied Health Sciences, Suan Sunandha Rajabhat University, Samut Songkhram 75000, Thailand.

出版信息

Parasite. 2025;32:37. doi: 10.1051/parasite/2025030. Epub 2025 Jun 24.

DOI:10.1051/parasite/2025030
PMID:40552732
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12187069/
Abstract

Leishmaniasis is one of the most important zoonotic diseases. Recently, Leishmania (Mundinia) martiniquensis, Leishmania (Mundinia) orientalis, and Leishmania (Mundinia) chancei have been reported as new human pathogens. Trypanosomatids, apart from Leishmania spp., such as Crithidia spp., are also occasionally capable of infecting humans. Here, a one-step multiplex qPCR assay for the simultaneous identification and quantification of L. martiniquensis and L. orientalis/L. chancei and detection and quantification of trypanosomatids was developed using ITS1 as the molecular target and human RNase P as the internal control gene. The assay was evaluated using 44 positive residual DNA samples from leishmaniasis patients and 25 negative DNA samples. Results revealed that the limits of detection (LOD) of the assay for L. martiniquensis, L. orientalis, and Crithidia sp. (CLA-KP1) were 1.699 (0.0255), 1.717 (0.0292), and 1.763 (0.0882) fg/reaction (parasite equivalents/reaction), respectively. The assay had high analytical specificity. The mean Cq values of the intra-assays and inter-assays differed by less than 1, indicating the reliability of the assay. Evaluation results revealed that the assay could identify L. martiniquensis and L. orientalis in clinical samples with 100% sensitivity and 100% specificity. In conclusion, the ITS1/human RNase P multiplex qPCR assay offers a rapid and reliable diagnostic tool for identifying and quantifying L. martiniquensis and L. orientalis/L. chancei and detecting and quantifying trypanosomatids in clinical samples within a single reaction. This assay provides an advancement in the diagnostic capabilities for leishmaniasis and trypanosomatid infections, potentially improving patient management and surveillance efforts.

摘要

利什曼病是最重要的人畜共患病之一。最近，马提尼克利什曼原虫（蒙迪尼亚利什曼原虫）、东方利什曼原虫（蒙迪尼亚利什曼原虫）和钱氏利什曼原虫（蒙迪尼亚利什曼原虫）已被报道为新的人类病原体。除利什曼原虫属外，锥虫科的其他属，如克氏锥虫属，也偶尔能够感染人类。在此，开发了一种一步多重qPCR检测方法，以ITS1作为分子靶点，人核糖核酸酶P作为内参基因，用于同时鉴定和定量马提尼克利什曼原虫和东方利什曼原虫/钱氏利什曼原虫，并检测和定量锥虫科。使用来自利什曼病患者的44份阳性残留DNA样本和25份阴性DNA样本对该检测方法进行了评估。结果显示，该检测方法对马提尼克利什曼原虫、东方利什曼原虫和克氏锥虫（CLA-KP1）的检测限分别为1.699（0.0255）、1.717（0.0292）和1.763（0.0882）fg/反应（寄生虫当量/反应）。该检测方法具有较高的分析特异性。批内和批间的平均Cq值差异小于１，表明该检测方法具有可靠性。评估结果显示，该检测方法能够以100%的灵敏度和10０%的特异性鉴定临床样本中的马提尼克利什曼原虫和东方利什曼原虫。总之，ITS1/人核糖核酸酶P多重qPCR检测方法提供了一种快速可靠的诊断工具，可在单一反应中鉴定和定量马提尼克利什曼原虫和东方利什曼原虫/钱氏利什曼原虫，并检测和定量临床样本中的锥虫科。该检测方法提高了利什曼病和锥虫感染的诊断能力，可能改善患者管理和监测工作。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/5e9d9775f91a/parasite-32-37-fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/0eef67ec5bdb/parasite-32-37-fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/a9caaa964056/parasite-32-37-fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/5175cf5a1cd3/parasite-32-37-fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/160a81c260ca/parasite-32-37-fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/e6148df6c087/parasite-32-37-fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/08234ea48695/parasite-32-37-fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/5e9d9775f91a/parasite-32-37-fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/0eef67ec5bdb/parasite-32-37-fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/a9caaa964056/parasite-32-37-fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/5175cf5a1cd3/parasite-32-37-fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/160a81c260ca/parasite-32-37-fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/e6148df6c087/parasite-32-37-fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/08234ea48695/parasite-32-37-fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46df/12187069/5e9d9775f91a/parasite-32-37-fig7.jpg

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本文引用的文献

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Curr Res Parasitol Vector Borne Dis. 2025-3-18

[2]
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Curr Res Parasitol Vector Borne Dis. 2024-9-24

[3]
Incidence and persistence of asymptomatic Leishmania infection among HIV-infected patients in Trang province, Southern Thailand: A cohort study.

PLoS Negl Trop Dis. 2024-10

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Am J Trop Med Hyg. 2024-10-2

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PLoS Negl Trop Dis. 2024-7

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PLoS Negl Trop Dis. 2024-1

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Sci Rep. 2023-12-28

[8]
Genetic characterization and description of Leishmania (Leishmania) ellisi sp. nov.: a new human-infecting species from the USA.

Parasitol Res. 2023-12-15

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Receiver operating characteristic curve analysis in diagnostic accuracy studies: A guide to interpreting the area under the curve value.

Turk J Emerg Med. 2023-10-3

[10]
Clinical performance of a quantitative pan-genus Real-time PCR assay for diagnosis of cutaneous and visceral leishmaniasis.

Pract Lab Med. 2023-10-5

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