Study On The Molecular Diagnosis System Of Group A Enterovirus Using Multiple Real-time PCR And RT-LAMP

BackgroundHand,foot and mouth disease（HFMD）is a worldwide infectious disease in children less than 5 years old.In 2008,it ranked the first in category C infectious diseases in China.Currently,the major pathogens HFMD include EV71,CVA16,CVA6 and CVA10.Except EV71,no vaccines or effective antivirals are available.Therefore,the establishment of rapid,simple,sensitive and specific laboratory testing methods for clinical diagnosis and typing would provide a reliable basis for personalized clinical treatment,and also for the prevention and control of HFMD epidemics.ObjectiveThe purpose of this project is to establish a rapid and accurate detection system for major group A eneroviruses（EV71,CVA16,CVA6 and CVA10）based on the TaqMan probe multiplex fluorescence quantitative RT-PCR method and the RT-LAMP method.Methods1.Amplication of the VP1 genes and the primer designThe VP1 gene sequences of EV71,CVA16,CVA6 and CVA10 were downloaded from GenBank and compared with each other,respectively.Primers and probes were designed in the conserved regions.The VP1 primers of each serotype were used to amplify the target fragment.2.Preparation of the standard2.1 Preparation of the standard for multiple real-time PCR assaysThe amplified target gene fragment was purified,ligated to the T vector,and transformed into E.coli.Finally,the plasmid was extracted and the standard was prepared after 10-fold dilution.2.2 Preparation of the standard for the RT-LAMP assaySimilarly,the amplified target gene fragment was purified,ligated to the T vector,and transformed into E.coli.Finally,the plasmid was extracted and was transcribed into RNA using an in vitro transcription kit.After purification,10-fold gradient dilutions were prepared as the standard.3.Optimization of the reaction systems3.1 Optimization of the multiple real-time PCR reaction systemsThe reaction systems and reaction conditions of four major enteroviruses were optimized,including the reaction temperature,the concentration of primers and probes,to explore the best reaction parameters.3.2 Optimization of the RT-LAMP reaction systemsThe RT-LAMP reaction systems and reaction conditions of enterovirus CVA6 and CVA16 were optimized,including betaine concentration,dNTP concentration,Mg²⁺concentration,SYBR Green I concentration,calcein concentration and optimum temperature,to determine the best RT-LAMP reaction parameters.4.Specificity,sensitivity and reproducibility of multiple real-time PCR and RT-LAMP assaysThe designed primers and probes were used to detect each type of enterovirus isolated in our laboratory to verify the specificity of the multiple real-time PCR and RT-LAMP assays.Standard curves were constructed for each type of enterovirus using the standards obtained in the previous step.The sensitivity and repeatability of each assay was also tested again.Statistical analyses were performed using the SPSS program.5.Performance of the assays5.1 Performance of the Multiple real-time PCR assasy to detect clinical samplesClinical samples were tested to determine the performance of the multiplex fluorescence quantitative PCR system established in the current study.Total RNAs were extracted from the clinical samples using Trizol,which were reversely transcribed into cDNA.Single fluorescence quantitative PCR,multiplex fluorescence quantitative PCR and enterovirus universal quantitative PCR were used to detect these samples,respectively.The positive samples were sequenced by Sanger sequencing to verify the performance of the multiplex real-time PCR assay.5.2 Performance of the RT-LAMP assay to detect clinical samples97 clinical samples were tested to determine the performance of the RT-LAMP assays established for CVA6 and CVA16,respectively.Total RNAs were extracted from clinical samples using Trizol and were then detected using RT-LAMP and PCR,respectively.Positive samples were then sequenced by Sanger sequencing to verify the performance of the RT-LAMP assay.Results1.Our designed primers and probes only showed amplification curve in the corresponding template’s channel,indicating high specificity of the multiplex fluorescence quantitative PCR established in the present study.The sensitivity test showed that the detection limits of the assay for EV71,CVA16,CVA6,and CVA10 were 3×10³ copies/μl,5×10² copies/μl,4×10³ copies/μl and 4×10¹ copies/μl,respectively.The performance of the multiple PCR assay was evaluated using clinical samples.The universal quantitative real-time PCR identified 73 enterovirus positive samples,single quantitative PCR identified 82 positive samples,and multiple quantitative PCR identified 76 positive samples.Further Sanger sequencing proved that the serotypes of all the 76 positive samples were correctly identified by our multiple quantitative PCR assay.2.The RT-LAMP detection systems for CVA6 and CVA16 were also successfully established in this study,respectively.These assays had high specificity and the amplification curve only appeared in the corresponding reaction channel.The detection limits for CVA6 and CVA16 were up to 2.0×10³ copies/μl and 3.0×10² copies/μl,respectively.In addition,calcein dye was added as a reaction indicator before amplification leading to a green color for positives which could be easily inspected by eyes.A total of 97clinical samples were detected by RT-LAMP and RT-PCR,respectively.The RT-LAMP results showed that there were 49 CVA6 positive samples and 23 CVA16 positive samples,all of which were further verified by Sanger sequencing.Therefore,the established RT-LAMP assays had high accuracy when detecting clinical samples.Conclusions1.A multiplex fluorescence quantitative PCR detection system for enterovirus EV71,CVA16,CVA10 and CVA6 was successfully established in this study,with good specificity,high sensitivity and high throughput.2.RT-LAMP detection methods for enterovirus CVA6 and CVA16 were also successfully established in the present study.The method is characterized by high specificity,high sensitivity,simple,and easy popularization,without using complicated and expensive instruments.