Molecular Epidemiological Features Of Coxackievirus A16 In Shenzhen And Establishment Of RT-LAMP

Background and objectiveHand, foot, and mouth disease (HFMD) is a common infectious disease of young children associated with infection of Enteroviruses, this disease has high infections and complex transmission route which could course large outbreaks in a short time. Coxsackievirus A16 (CA16) and human Enterovirus 71 (HEV71) are major causative agents of this disease. HFMD is typically mild and self-limiting, followed by developing vesicular lesions on hands, feet, mouths, and buttocks, severe cases usually presented with neurologic complications including death. EV71-associated HFMD are more severe than the disease caused by CA16 so that little attention was paid to CA16 strains, few studies was fully described CA16 molecular characteristics and no unified standard was used to divide genotype. CA16 possibly has a higher percentage than it actually is considering it's mild during the epidemic of HFMD. In recent years, fatal CA16 infection has also been described in children who had HFMD associated with myocarditis and in an adult with pneumonitis. Therefore, the epidemiological investigation and gene characteristics of CA16 should be determined. This study was conducted to investigate the casual causes of CA16 in HFMD patients during five-year study in Shenzhen, China from 2005 to 2009. To sequence the VP1 gene of CA16 isolated, it'll be analyzed the nucleotide identity with other strains at home and abroad. At the same time it will provide information on continuously monitor epidemiological and molecular evolution of CA16, and a high degree of vigilance should be maintained over the disease situation.There are many conventional methods to detect CA16 in HFMD, such as virus isolation and cultivation, immunohistochemistry, neutralization test and reverse transcription-polymerase chain reaction (RT-PCR) etc. Classic cell separation cultivation techniques and specific antibody test method have cumbersome steps and long cycle, and some enteroviruses are difficult to cultivate in the cell lines, which easilly lead to some viruses are missed. Molecular biology diagnostic techniques based on the nucleic acid amplification such as PCR, RT-PCR and real-time fluorescent RT-PCR play a significant role in detection of pathogenic micro-organisms and disease diagnosis. Although these techniques are highly sensitive, they are not suitable for evaluation in field situation due to the involvement of complex procedures, sophisticated and expensive laboratory equipments, numerous trained workers and the application of toxic reagents such as ethidium bromide. Therefore, this developed RT-LAMP technology can provide rapid and sensitive diagnosis of CA16, which can be ideally applied in situ without transferring samples to central laboratory.Loop-mediated isothermal amplification (LAMP) was established as a new nucleic acid amplification method by Notomi in 2000, This method used Bst DNA polymerase large fragment and four or six specially designed primers that target six or eight independent regions on DNA sequence to accomplish auto-cycling strand displacement DNA synthesis. Reaction results can be amplified by product of the precipitation of magnesium pyrophosphate to determine turbidity, also can be added SYBR Green I, ethidium bromide (EB) or other nucleic acid dyes for coloring to watch. The advantage is specificity, amplification efficiency, responsive and easy to operate. The technology has been applied to a variety of bacterial and viral gene detection by domestic and foreign scholars because of its specificity, amplification efficiency, responsive and easy to operate. The report about the technology used in detection of CA16 gene has not yet retrieved. In this paper, two pairs of specific primers were designed to capsid protein VP1 gene sequence of CA16. RT-LAMP amplification system was optimized for the detection of the target sequences, and its specificity and sensitivity were verified through experiments. The examinations have carried out to detect CA16 gene in the specimens of clinical diagnosis HFMD Patient through this technology in Shenzhen city.The result of RT-LAMP was compared with the result of reverse transcription-Polymerase chain reaction (RT-PCR) and fluorescence quantitative RT-PCR (Real-time RT-PCR) methods. The purpose is to seek fast, simple and practical new methods that applied for clinical diagnosis of CA16 infection.Methods1. Molecular epidemiology characteristics of CA16CA16 positive strains were diagnosed by real-time RT-PCR to conduct further genetic analysis. A total of 69 CA16 were selected to amplify the whole VP1 region by RT-PCR techniques, using specific primers. A phylogenetic tree was constructed by comparison of the sequences with all subgenotypes of CA16 using DNASTAR and Mega 4.1 software.2. Establishment of RT-LAMP for detection CA16VP1 nucleotide sequences were useful for describing different genotypes of CA16 strains. The complete CA16 VP1 sequences of all genotypes were available in the Gene Bank database and aligned using CLUST X software to identify conserved regions. The VP1 sequence were analyzed by RT-LAMP primer design software and several sets of four specific primers were automatically designed. To optimize the reaction condition of RT-LAMP, and then to confirm sensitivity, specificity and repeatability of optimization RT-LAMP. Finally, evaluate RT-LAMP assay using clinical samples.Results1. Analysis molecular epidemiology characteristics of CA16A total of 1,161 cases of HFMD were collected by the Center for Disease Control and Prevention in Shenzhen from 2005 to 2009, CA16 was detected in the samples collected in each year, and total positive rate of CA16 strains during real-time RT-PCR was 21.4%. The annual detection rates were 27.0%,51.6%,27.6%, 17.8%and 17.0%, respectively. The data revealed that a majority of these patients (92.7%) were less than 5 years old, no statistically significant difference was observed between boys and girls in total incidence rate of CA16 infection in the period of five years (P=0.469). Overall, monthly detection suggested that two peaks were associated with CA16 infection. The first peak occurred from March to June and the second peak was evident from September to November. Meanwhile, the first peak was consistently higher than the second. Most patients were concentrated in Longgang and Baoan and less cases in Futian, Nanshan, Luohu and Yantian.The mean nucleotide and amino acid homology of 69 CA16 isolated from Shenzhen were 96.8% and 99.9%, respectively. In order to further determine the molecular epidemiology and genotype of Shenzhen CA16 strains, a phylogenetic dendrogram was constructed with 25 CA16 strains collected from Shenzhen and 37 genotype-identified CA16 sequences from other Chinese locations or other countries, according to similar genotypes standard of EV71, a difference of at least 15% in the VP1 gene was used to distinguish genotypes, CA16 strains were divide into two different major clusters, genotypes A and B, genotype B could be chronologically divided into B1 and B2 subtypes, and subtype B2 could be further divided into B2a, B2b and B2c. Genotype B and C in previous studies should be combined into genotype B. Genotype B in their study was considered as B1 Subtype, and genotype B was considered as B2 in our study. Amino acid evolution in the VP1 region was small, even though higher activity on the same subtypes than different ones was exhibited. All CA16 strains detected in this study belonged to cluster B2a (66.7%)and B2b (33.3%). Thus, recent genotypes of CA16 in Shenzhen were similar to those detected in other Chinese provinces and countries.2. RT-LAMP for detection CA16The CA16 RT-LAMP assay is fast, simple and low cost, the reaction could be finished in sixty minutes, which was less than that of conventional RT-PCR (90 min) and Real-time RT-PCR (20 min). The detection limit of this assay was 81 copies/tube, which was ten-fold higher in sensitivity than RT-PCR and equal to real-time RT-PCR. This assay aslo has high specificity and repeatability, and on false positive was found. A good correlation between RT-LAMP and real-time RT-PCR was observed on the basis of the analysis of clinical samples.Conclutions1. Total positive rate of CA16 strains was 21.4% from 2005 to 2009. CA16 is one of the main causative agents of HFMD in Shenzhen, high-risk group was children less than five years old, March to June and September to November are the epidemic season of this disease, suburban children are relatively more susceptibility to CA16-associated HFMD than urban children, no statistically significant difference was observed between boys and girls in incidence rate of CA16.2. CA16 strains were divide into genotypes A and B, genotype B could be divided into subtype B1 and subtype B2, and subtype B2 could be further divided into B2a, B2b and B2c.The homogeneity of nucleotide sequence and amino acid of CA16 strains circulating in those five years high in study. All CA16 belong to cluster B2a, also there was cluster B2b.3. In this paper, specific RT-LAMP primers were designed according to the conserved region of VP1 genome for the first time. A one-step, single-tube RT-LAMP assay was developed and validated for the detection of CA16.4. RT-LAMP method in this assay is 90 minutes faster than RT-PCR and 30 minutes faster than Real-time RT-PCR in reaction time, and is quadruple less than RT-PCR and twice less than Real-time RT-PCR in the test cost.5. A good correlation between the RT-LAMP and real-time PCR results was observed during the evaluation of clinical samples which were diagnosed as HFMD.