摘要
目的 建立一种可同时检测十二指肠贾第虫、微小隐孢子虫、毕氏肠微孢子虫和莫尼茨绦虫等4种山羊肠道寄生虫的多重PCR检测方法,并初步评估其检测效能.方法 基于GenBank中十二指肠贾第虫(GenBank登录号:XM_001710026.2)、微小隐孢子虫(GenBank登录号:XM_626998.1)、毕氏肠微孢子虫(GenBank登录号:KJ719492.1)和莫尼茨绦虫(GenBank登录号:OM296991.1)相应基因的保守序列设计4对特异性引物,建立并优化可同时检测上述4种寄生虫的多重PCR方法.2022年10-12月,从广东省湛江市4个山羊养殖场采集116份新鲜山羊粪便样本,其中96份用于所建立多重PCR方法的检测效能评价、20份作为样本检测的本底对照.分别采用单病原PCR检测方法及本研究所建立的多重PCR方法,对96份山羊粪便DNA样本进行检测.以单病原PCR检测结果为金标准,计算多重PCR法的检测灵敏度、特异度、阳性预测值和阴性预测值.结果 本研究所建立的多重PCR方法可同时扩增出十二指肠贾第虫、微小隐孢子虫、毕氏肠微孢子虫和莫尼茨绦虫特异性基因片段,其大小分别为1 400、755、314 bp和585 bp,检出限为≥102 拷贝数的病原DNA克隆质粒;该方法对日本血吸虫、羊前后盘吸虫、细粒棘球绦虫、芽囊原虫和平腹吸虫基因扩增结果均为阴性.采用单病原PCR和所建立的多重PCR方法检测96份山羊粪便DNA样本,40份(40/96,41.67%)粪便DNA经单病原PCR检测出现阳性扩增产物,其中39份经多重PCR检测亦出现阳性扩增产物,平均符合率为97.50%(39/40).96份样本中,多重PCR方法分别检出十二指肠贾第虫、微小隐孢子虫、毕氏肠微孢子虫、莫尼茨绦虫感染阳性26(27.10%)、22(22.90%)、24(25.00%)、9份(9.40%),与单病原PCR检测结果一致.以单病原PCR检测结果为金标准,多重PCR法对山羊粪便样本中十二指肠贾第虫、毕氏肠微孢子虫、微小隐孢子虫和莫尼茨绦虫DNA检测灵敏度分别为96.15%、95.83%、100.00%、100.00%,阴性预测值分别为98.90%、98.92%、100.00%、100.00%,阳性预测值均为100.00%.结论 本研究建立了一种可同时检测十二指肠贾第虫、微小隐孢子虫、毕氏肠微孢子虫和莫尼茨绦虫等4种山羊常见寄生虫的多重PCR方法,该方法灵敏度高、特异性好,适用于大规模羊群粪便样本快速筛查.
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 ef-ficiency.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.bi-eneusi(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,specifici-ty,positive predictive value,and negative predictive value of the multiplex PCR assay were evaluated for detection of G.duodena-lis,C.parvum,E.bieneusi and Moniezia DNA in goat stool samples with the single-target PCR assay as the gold standard.Re-sults The multiplex PCR assay developed in this study allowed simultaneous amplification of specific gene fragments of G.duo-denalis,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 Schistoso-ma 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 sin-gle-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 predic-tive 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.bi-eneusi and Moniezia DNA in goat stool samples,respectively,if the single-target PCR assay served as the gold standard.Conclu-sion 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.
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