Four Fluorescent Quantitative Realtime RT-PCR Assay For Detection Of Coxsacki-Evirus And Universal Enterovirus

Coxsackie virus, one type of enteroviruses, is divided into A and B serotype, the group B has six subtype. B serotype Coxsackie virus can cause respiratory infections, diarrhea, meningitis, myocarditis, severe neonatal infection, et al, especially the most common cause of viral myocarditis (VMC) is coxsackie viral infection. Viral myocarditis may also be caused by other enterovirus. Current clinical diagnosis of viral myocarditis, mainly relies on clinical symptoms and routine biochemical tests and electrocardiograms. However, due to the. symptoms of the onset viral myocarditis are not typical.It is often involved myocardial when clinical symptoms appears, causing irreversible myocardial damage, resulting in serious consequences. The serological method has been widely used in diagnosis of viral infections, but can not confirm the species of infection virus. In this research, the conserved sequence of the the coxsackie virus B1, B2, B3and other enterovirus were analyzed, highly specific primers and Taqman probe were designd respectively, and set up a multiplex real-time RT-PCR method. The one-step method can detect CoxB1, CoxB2, CoxB3and other enterovirus simultaneously. This study includes2parts, the first part is the construction of a multiplex real-time RT-PCR method for detecting coxsackie virus B1, B2, B3, and other enterovirus; the second part is the clinical application of multiplex real-time RT-PCR method. Part one:construction of a multiplex real-time RT-PCR method for detecting coxsackie virus B1, B2, B3, and ther enterovirusLMethods(1)Downloaded multiple gene sequences about CoxB1, CoxB2, CoxB3and other enterovirus from NCBI. using DNAMAN software to compare their similarity and identified conserved regions of the viral genome mentioned above. The highly specific primers and Taqman probes of the conservative regions in viral genome were designed by Primer Express3.0software, Primers and the probes sequence are validated by Blast.(2)Optimization of reaction system, using nucleic acid fragments of CoxB1, CoxB2, CoxB3and isolates (Such as rotavirus, norovirus, Sapovirus, adenovirus, EV71, CoxA16) to evaluate the specificity of the method, the number of copies that have been calibrated standards were diluted, and fluorescent PCR reactions performed in parallel to compare the sensitivity.(3)Furthermore, each concentration of diluted positive nucleic acid were detected6times reduplicatively, and get the CT value to calculate the standard deviation and coefficient of variation, verify the repeatability of the method.2.Results(1)Sensitivity and Specificity The sensitivity of the method to detect CoxB1, CoxB2, CoxB3and other enteroviruses has reached103copies/ml. This method has a high specificity for CoxB1, CoxB2, CoxB3and other enterovirus, the primer probe of CoxB1、CoxB2、CoxB3have no cross-reactivity with other viruses, such as rotavirus, norovirus, Sapovirus adenovirus, EV71, CoxA16.(2)Repeatability For six duplicate detection of each concentration samples, the results of different nucleic acid concentration of the Ct value standard deviation is between0.09-0.28, the coefficient of variation were less than1.0%, with good repeatability.3.ConclusionWe successfully constructed a rapid and accurate multiplex real-time RT-PCR method, this method can simultaneously detect and distinguish CoxB1, CoxB2, CoxB3and other unclassified enterovirus, therefore this method is worthy for extending in clinic.Part two:the clinical application of multiplex real-time RT-PCR method for detecting coxsackie virus Bl, B2, B3, and other enterovirus1.MethodsUsing multiplex real-time RT-PCR method for detecting535children with viral myocarditis feces after treatment and43pharyngeal swab specimens before treatment, statistical analysis of the results. And randomly selected10positive samples were sequenced, and further verify the accuracy of the method.2.Results578specimens were detected enterovirus153, positive rate was26.5%(153/578), including CoxB1virus69, the positive rate was11.9%(69/578); including CoxB2virus18, the positive rate was3.1%(18/578); including CoxB3virus27, the positive rate was4.7%(27/578); other types of enterovirus39, the positive rate was6.7%(39/578). Further sequencing, positive specimens test result were consistent with the test results assay by this method.3. ConclusionWe successfully constructed a simple and efficient, reproducible multiplex real-time RT-PCR method, this method can provide early diagnosis for clinically suspected intestinal virus infection of children, especially children with infantile viral myocarditis, and can distinguish the types of virus infection and titer, to provide the reference for the clinical treatment.