Development and preliminary application of a multiplex PCR assay for simultaneous detection of four intestinal parasites in goats
10.16250/j.32.1374.2024046
- VernacularTitle:一种同时检测4种山羊肠道寄生虫多重PCR方法的 建立与初步应用
- Author:
Yilong LI
1
,
2
;
Xuanru MU
1
,
2
;
Hui XU
1
;
Xiaoping LUO
3
;
Runzi YU
1
;
Xinyi XU
1
;
Linlin YANG
1
;
Xingang YU
1
;
Yang HONG
4
,
5
Author Information
1. School of Life Science and Engineering, Foshan University, Foshan, Guangdong 528231, China
2. Co-first authors
3. Veterinary Research Institute,Inner Mongolia Academy of Agricultural & Animal Husbandry Sciences, China
4. National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research), National Health Commission Key Laboratory of Parasite and Vector Biology, Shanghai 200025, China
5. Hainan Tropical Diseases Research Center (Hainan Sub-Center,Chinese Center for Tropical Diseases Research), Haikou, Hainan 571199, China
- Publication Type:Journal Article
- Keywords:
Intestinal parasite;
Giardia duodenalis;
Cryptosporidium parvum;
Enterocytozoon bieneusi;
Moniezia;
Multiplex PCR;
Goat
- From:
Chinese Journal of Schistosomiasis Control
2024;36(4):376-383
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a multiplex PCR assay for simultaneous detection of four intestinal parasites, including Giardia duodenalis, Cryptosporidium parvum, Enterocytozoon bieneusi and Moniezia, and to preliminarily evaluate its detection efficiency. Methods Four pairs of specific primers were designed based on the conserved sequences of the corresponding genes of G. duodenalis (GenBank accession number: XM_001710026.2), C. parvum (GenBank accession number: XM_626998.1), E. bieneusi (GenBank accession number: KJ719492.1) and Moniezia (GenBank accession number: OM296991.1) retrieved from the GenBank database, and a multiplex PCR assay for simultaneous detection of G. duodenalis, C. parvum, E. bieneusi and Moniezia was developed and optimized. A total of 116 fresh goat stool samples were collected from four goat farms in Zhanjiang City, Guangdong Province during the period from October to December 2022, including 96 samples used for evaluating the detection efficacy of the multiplex PCR assay, and 20 samples as baseline controls for sample testing. Genomic DNA extracted from 96 goat stool samples was tested using the single-target PCR assay and the developed multiplex PCR assay, and the sensitivity, specificity, positive predictive value, and negative predictive value of the multiplex PCR assay were evaluated for detection of G. duodenalis, C. parvum, E. bieneusi and Moniezia DNA in goat stool samples with the single-target PCR assay as the gold standard. Results The multiplex PCR assay developed in this study allowed simultaneous amplification of specific gene fragments of G. duodenalis, C. parvum, E. bieneusi and Moniezia, with 1 400, 755, 314 bp and 585 bp in sizes, respectively, and the detection limit was 102 and higher copies of parasite DNA clones, while the multiplex PCR assay was negative for gene amplification of Schistosoma japonicum, Fasciola hepatica, Echinococcus granulosus, Blastocystis hominis and Homalogaster paloniae. Single-target PCR assay and the developed multiplex PCR assay were employed to test DNA samples extracted from 96 goat stool samples, and single-target PCR assay tested positive in 40 goat stool samples (41.67%), including 39 positive samples tested with the multiplex PCR assay, with a mean coincidence rate of 97.50% (39/40). The multiplex PCR assay tested positive for G. duodenalis DNA in 26 goat stool samples (27.10%), C. parvum DNA in 22 samples (22.90%), E. bieneusi DNA in 24 samples (25.00%), and Moniezia in 9 samples (9.40%), which was consistent with the detection using the single-target PCR assay. The sensitivity, negative predictive value, and positive predictive value of the multiplex PCR assay were 96.15%, 95.83%, 100.00% and 100.00%, 98.90%, 98.92%, 100.00% and 100.00%, 100.00%, 100.00%, 100.00% and 100.00% for detection of G. duodenalis, C. parvum, E. bieneusi and Moniezia DNA in goat stool samples, respectively, if the single-target PCR assay served as the gold standard. Conclusion A highly sensitive and specific multiplex PCR assay has been developed for simultaneous detection of G. duodenalis, C. parvum, E. bieneusi and Moniezia in goats, which is suitable for rapid, large-scale screening of intestinal parasites in sheep stool samples.
