Double Fluorescent Quantitative RT-PCR Assay ForCVA2/CVA5 Or CVA6/CVA10 Detection Of Enterovirus

Objective:Hand, foot and mouth disease (HFMD) is a kind of infectious disease which is commonly occurred in children caused by human enterovirus (HEV). Up to now, more than 90 genotypes of HEV viruses have been reported, and divided into 4 groups including A, B, C, D, among which human enterovirus type71 (EV71) belonging to HEV-A and human coxsackie virus type A16 (CAV16) have been the most important pathogens. However, increased research reports show some other serotypes of HEV such as CVA2, CVA5, CVA6, CVA10, etc, also as the common causative pathogen of HFMD. Currently, many countries and regions broke out in CVA2, CVA5, CVA6, CVA10 type-based foot and mouth disease, and Zhejiang has no relevant reports. CVA2, CVA5, CVA6, CVA10 type four pathogen detection nor relatively timely, efficient, accurate and convenient test kit. In the present HEMD pathogen diagnosis market, HFMD is normally determined by detecting the universal types of enterovirus, EV71 or CVA16 that usually cause the uncertainty or missing of the pathogens, which have a negative effect on the patients such as delays in diagnosis, also cause waste of reagents. Therefore, the development of diagnosis technology for detecting HFMD caused by CVA2, CVA5, CVA6, CVA10, seems urgent. In this study, in order to identify and determine the HFMD caused by CVA2, CVA5, CVA6, CVA10, rapidly and accurately, double real-time fluorescent quantitative PCR was performed to simultaneously detect four types of coxsackie viruses including CVA2, CVA5, CVA6 and CVA10 and used for the further clinical application.Part one construction of a double real-time RT-PCR methodfor detecting coxsackie virus A2/A5 or coxsackie virus A6/A10Methods:1. Multiple gene sequences about coxsackie virus A2, A5 and coxsackie virus A6, A10 were downloaded from NCBI. DNAMANs oftware was then used for comparing their similarity and identifying conserved regions of the viral genome mentioned above. The highly specificprimers and Taqman probes of the conservative regions in viral genome were designed by Primer Express 3.0 software. Primers and the probes sequence were validated by Blast.2. By optimization of reaction system, nucleic acid fragments of coxsackie virus A2, A5 and coxsackie virus A6, A10 and isolates(Such as Ev71, CoxA16, CoxB 1, CoxB3, ECHO virus, influenza A virus, respiratory syncytial virus, Bocavirus) were used to evaluate the specificity of the method. The number of copies calibrated standards were diluted, and fluorescent PCR reactions performed in parallel to compare the sensitivity. Each concentration of dilutedpositive nucleic acid was detected 6 times, and the CT value was acquired for calculating the standard deviation and coefficient of variation to verify the repeatability of the method.Results:1. Optimization of reaction system,double real-time RT-PCR method had a high specificity for coxsackie virus A2, A5, the primer probe of coxsackie virus A2, A5 had no cross-reactivity with other viruses, such as coxsackie virus A6,coxsackie virus A10, Ev71, CoxA16, CoxB 1, CoxB3, ECHO virus, influenza A virus, respiratory syncytial virus, Bocavirus. The sensitivity of the method to detect oxsackie virus A2, A5 has reached 102. Repeatability for three duplicate detection of each concentration samples, theresult of different nucleic acid concentration of the Ct value standard deviation was between0.13～0.28, the coefficient of variation was less 1.49%,with good repeatability.2. Optimization of reaction system, double real-time RT-PCR method had a high specificity for coxsackie virus A6, A10, the primer probe of coxsackie virus A6, A10 had no cross-reactivity with other viruses, such as coxsackie virus A2,coxsackie virus A5, Ev71, CoxA16, CoxB 1, CoxB3, ECHO virus, influenza A virus, respiratory syncytial virus, Bocavirus. The sensitivity of the method to detect oxsackie virus A6, A10 had reached 102. Repeatability for three duplicate detection of each concentration samples, the result of different nucleic acid concentration of the Ct value standard deviation was between0.14～0.28, the coefficient of variation was less than 1.44%, with good repeatability.Conclusion:We successfully established a double real-time fluorescent quantitative PCR method for simultaneously detecting and identifying CVA2 and CVA5 in a single tube, also CVA6 and CVA10 in a single tube. The method showed the advantage of amplification in a closed single tube, simple and rapid response, well repeatability, real-time quantification and poor contamination, etc, and provided an accurate scientific basis for the clinical diagnosis, as being an effective detective technique.Part two Clinical application of double real-time RT-PCR methodfor detecting coxsackie virus A2/A5 or coxsackie virus A6/A10Methods:367 stool specimens were collected from the confirmed and suspected HFMD patients treated in 3 hospitals:The First Affiliated Hospital, School of Medicine, Zhejiang University, The children’s hospital, Zhejiang University School of Medicine, The Central Hospital of Huzhou, between March 2013 and June 2013 for the detection of CVA2 and CVA5. Another 419 stool specimens were collected from the confirmed and suspected HFMD patients treated in 3 hospitals:The First Affiliated Hospital, School of Medicine, Zhejiang University, The children’s hospital, Zhejiang University School of Medicine, The Central Hospital of Huzhou, between March 2013 and September 2013 for the detection of CVA6 and CVA10. Statistical analysis was then performed.10CVA2 and CVA5 positive strains were randomly selected for sequencing. Relative sequence results were compared with the reference nucleotide sequences from GenBank by Blast in the NCBI Database to determine the genotype of coxsackie virus. Evolutionary analysis was performed by using MEGA 4.0 software, genetic distance was calculated by Kimura-2 parameter Model. Phylogenetic tree was constructed by neighbor-joining (NJ) method. Homologous analysis was performed by using Vector NTI AlignX in BioEdit software.Results:The specimens were detected by the established RT-PCR method, and the results were as below following:Of 367 stool specimens,23 CVA2 positive specimens were detected with the positive rate of 6.3%,11 were CVA5 with the positive rate of 3.0%. Of 419 stool specimens,171 CVA6 positive specimens were detected with the positive rate of 40.8%,27 were CVA10 with the positive rate of 6.4%. The positive detection result was consistent with the results by sequencing. The VP1 gene of CVA2 strain showed high identity with the VP1 gene of CVA5 strain. The identity rate of the VP1 gene of CVA2 strain was between 92.3%-99% compared with the strains in neighboring countries or regions. Similarly, the identity rate of the VP1 gene of CVA5 strain was between 92%-97% compared with the strains in neighboring countries or regions, hand, Foot and mouth diseasecaused by CVA2, CVA5, CVA6, CVA10 in between 2012 and 2013, pathogen showed a trend of the pandemic, close genetic relationship and no variation with the migration.Conclusion:Our method could simultaneously detectCVA2 and CVA5, CVA6 and CVA10, respectively, and determine the titers of pathogens, thus reduce the uncertainty or missing of the pathogens and the wasted of diagnosis reagents, etc. This method was also with accurate quantification, and provided the reference basis forclinical treatment. Besides, this method played an important role in indicating the transmission of virus, and assisted in research on epidemiology of the pathogen.