Establishment And Application Of Multiplex Fluorescent Quantitative PCR For Detection Of Three Species Of Paragonimus Metacercariae

Objective:A specific detection method was established for the metacercaria detection of Paragonimus westermani,Paragonimus skrjabini and Euparagonimus cenocopiosus by the fluorescence Quantitative PCR,as well as the specific detection method was preliminarily evaluated by detection metacercariae from 4 provinces in China.Methods:1.The CO1 sequences of P.westermani,P.skrjabini,and E.cenocopiosus were downloaded respectively from GenBankTM for aligning and analyzing,and three pairs of specific primers and probes were designed to specifically identify the above three Paragonimus species.Standard positive plasmids were constructed by PCR amplification using specific primers,and the standard positive plasmid sequencing results were compared with published Paragonimus sequences in GenbankTM.2.A single fluorescent quantitative PCR assay for P.westermaniP.skrjabiniand E.cenocopiosus was established by the optimizing concentration primer,probe concentration,and annealing temperature for the three Paragonimus species.The specificity test,sensitivity test and corresponding standard curve were also carried out.3.The multiplex fluorescent quantitative PCR method for simultaneous identification and detection of P.westermani P.skrjabini and E.cenocopiosus in a single tube was established using primers and probes with the same sequence as the single fluorescent quantitative PCR assay and optimizing the concentration.Moreover,specificity tests,sensitivity tests,and standard curves were performed.4.Paragonimus metacercariae samples collected from Lantian Town,Xiuning County,Anhui Province,Doumen Town,Yongjia County,Zhejiang Province,Shaoyuan Town,Jiyuan City,Henan Province,Lingyao Town,Zhenghe County,Fujian Province,and Hexi Town,Nanjing County,Zhangzhou City,Fujian Province.The collected metacercariae samples were classified and identified simultaneously by conventional PCR and multiplex fluorescent quantitative PCR.The results can assess the classification identification ability of the multiplex fluorescent quantitative PCR method.Results:1.The constructed positive plasmids share 99.65%?98.17%?98.85%similarity with P.westermani(AY140692.1),P.skrjabini(AY618798.1),and E.cenococopiosus(AF 159594.1)published in GenBankTM,respectively.2.The established single fluorescent quantitative PCR method for P.westermani,P.skrjabini,as well as E.cenocopiosus had amplification curves only for the target gene and no specific amplification for other trematodes.In the sensitivity test,detecting the lowest concentrations of the three paragonimus single fluorescent quantitative PCR method were 30.5 copies/?L(P.westermani),3.21 copies/?L(P.skrjabini),and 3.22 copies/?L(E.cenocopiosus),respectively.The amplification efficiencies,amplification curves equation and correlation coefficient of the three paragonimus single fluorescent quantitative PCR method were the following:P.westermani E=96.6%,y=-6.8128x 37.059,R2?0.9998;P.skrjabini E=91.8%?Y=-7.0721x+45.106?R2=0.9997;E.cenocopiosus E=105.40%?Y=-6.3993x+33.957?R2=0.9995.It can be seen that the single fluorescent quantitative PCR method for the three Paragonimus species established in the study meets the basic parameters of fluorescent quantification,and has high specificity and sensitivity.3.The established multiplex fluorescent quantitative PCR method could specifically amplify the target fragment of P.westermani,P.skrjabini,E.cenocopiosus at the same time,while other parasites have no specific amplification.In the sensitivity test,detecting the lowest concentrations of the three species of Paragonimus multiplex fluorescence quantification PCR method were 3.05 × 102 copies/?L(P.westermani),3.21 × 103 copies/?L(P.skrjabini),and 3.22 × 102 copies/?L(E.cenococopiosus).The established standard curve equation,correlation coefficient,and amplification efficiency were as the following:P.westermani Y=3.7082x+27.501,R2=0.9984,E=85.8%;P.skrjabini Y=3.3715x+33.68,R2=0.9974,E=97.5%;E.cenocopiosus Y=3.5608x+18.874,R2=0.9938,E=93.0%.Therefore,the multiplex PCR assay established in this study meets the basic parameters of fluorescence quantificatio as well as with high specificity and sensitivity.4.Sample analysis results in 5 investigation sites detected by multiplex fluorescent quantitative PCR:Paragonimus metacercariae DNA from LanTian Town,Xiuning County,Anhui Province and Doumen Town,Yongjia County,Zhejiang Province showed positive results at FAM channel(465-510),while the others were negative(Fig2.4A).Paragonimus metacercariae DNA from Shaoyuan Town,Jiyuan City,Henan Province and Lingyao Town,Zhenghe County,Fujian Province showed positive results at the VIC channel(533-580),while others were negative(Fig2.4B).Paragonimus metacercariae DNA from Hexi Town,Nanjing County,Zhangzhou City,Fujian Province showed positive results at the CY5 channel(618-660),while the others were negative(Fig2.4C).The results showed that Paragonimus metacercariae from Xiuning County,Anhui Province and Yongjia County,Zhejiang Province were detected as Paragonimus westermani by multiplex fluorescent quantitative PCR,Paragonimus metacercariae from Jiyuan City,Henan Province and Zhenghe County,Fujian Province were P.skrjabini,and Paragonimus metacercariae in Zhangzhou City,Fujian Province were E cenococopiosus.5.Sixty-five metacercariae samples were classified and identified simultaneously by the multiplex fluorescent quantitative PCR and conventional PCR methods.The results showed that the taxonomic identification rate were 100%(65/65)by multiplex fluorescent quantitative PCR,while the taxonomic identification rate of Paragonimus at five survey sites by conventional PCR was 72.31%(47/65).it could be considered that the multiplex fluorescent quantitative PCR method was superior to the conventional PCR method.6.The positive rates of crabs at five survey sites were 82%(49/60)?41%(29/70)?55%(41/74)?65%(41/63)? 45%(32/71)in Lantian Town,Xiuning County,Anhui Province,Doumen Town,Yongjia County,Zhejiang Province,Shaoyuan Town,Jiyuan City,Henan Province,Lingyao Town,Zhenghe County,Fujian Province,and Hexi Town,Nanjing County,Zhangzhou City,Fujian Province,respectively.According to the morphological identification,the metacercariae species of Paragonimus in the five regions were P.westermani,P.westermani,P.skrjabini,P.skrjabini,and E.cenocopiosus,respectively.Molecular identification analysis after conventional PCR:the sequences of ITS2 and CO1 genes of Paragonimus metacercariae DNA from Xiuning County,Anhui Province and Yongjia County,Zhejiang Province were the most similar to those of P.westermani(KC417492.1,AF219379.2),ranging from 99%to 100%and 96%to 99%,respectively,and clustered into with P.westermani together.The sequences of ITS2 and CO1 genes of Paragonimus metacercariae DNA from Jiyuan City,Henan Province had the highest similarity with Paragonimus skrjabini genes(KX 129924.1,MK568551.1),ranging from 98%to 100%and 95%to 99%,respectively,all of which clustered into one branch with Paragonimus skrjabini.The sequence of the ITS2 gene of Paragonimus metacercariae DNA from Zhenghe County,Fujian Province had the highest similarity with the P.skrjabini gene(KX129924.1),ranging from 98%to 100%,and clustered into with P.skrjabini,Rmiyazaki together(two species branch is not obvious).However,the sequence of CO1 gene had the highest similarity(91%?94%)with those of P.miyazaki(AY618823.1,AY618834.1),clustered into a small branch with P.miyazaki together,but a large branch with P.skrjabini(the two parasite species clade was more obvious).The ITS2 gene sequence of Paragonimus metacercariae DNA from Zhangzhou City,Fujian Province had the highest similarity(100%)with the P.westermani gene(KC417492.1),the CO1 gene sequence had the highest similarity with the E.cenocopiosus gene(AF159595.1),ranging from 97%to 99%,and both the ITS2 gene sequence and the CO1 gene sequence clustered into with E.cenocopiosus together.The results showed that both morphological identification and molecular sequence analysis were consistent with the identification results of multiplex quantitative fluorescence PCR method,further demonstrating the high sensitivity and strong specificity of multiplex quantitative fluorescence PCR method.Conclusion:The established multiplex PCR method enables rapid identification of Paragonimus metacercariae samples in crabs.The establishment of this method provides technical support for the taxonomic study of Paragonimus and opens up a new path for the identification and detection method of Paragonimus metacercariae,which has important application value for the classification,geographical distribution,epidemiological investigation,food safety,and quarantine of Paragonimus.