Primary Study On Genetic Detection Of Enterovirus 71

Enterovirus71 (EV71), a positive-stranded RNA virus, belongs to the genus Enterovirus in the family Picornaviridae. The infection of Enterovirus 71 typically causes hand, foot and mouth disease or herpangina, followed by severe central nervous system complications, including aseptic meningitis, poliomyelitis-like paralysis, acute flaccid paralysis, Neurogena pulmonary edema and even death in some young children. The morbidity and mortality of Enterovirus 71 infection is significantly higher than that caused by other intestinal virues. For most patients, an infection with Enterovirus71 has no clinical symptom or only mild flu-like symptoms, which are often neglected and consequently result in more severe symptoms. Enterovirus 71 infections occur globally and have caused more than 10 large outbreaks worldwide. However, there is no effective vaccine or treatment strategy against an Enterovirus71 infection, so research on reagents for the early diagnosis of hand, foot and mouth disease has important clinical significance.Methods of laboratory diagnosis include virus isolation and culture, immunological methods, PCR technology etc. However, because of their low sensitivity, current techniques are neither large-scale nor high-throughput and thus early diagnosis is difficult. Immunological detection methods can not produce prompt and timely results and moreover have a low sensitivity, and therefore may delay the diagnosis.However, Real-time PCR and gene chip technology can be applied at the level of nucleicacids, which can detect the corresponding nucleic acids in a human body early, providing a rapid, efficient, sensitive and automated method for the early diagnosis of Enterovirus 71.In this paper, the cDNA subclonrd of Enterovirus 71 were constructed first, laying a foundation for further study. Second, the method of real-time PCR was established, analyzed and validated for its specificity, repeatability and sensitivity. Third, test probes of Enterovirus 71 were designed using software. The probes developed were used as a standard and an oligonucleotide microarray for Enterovirus 71 was developed.In the first part of this experiment, genes VP4, VP2, VP3 and VP1 of EV71 were amplified by long-fragment RT-PCR. Then subclones were constructed. Since Enterovirus71 is RNA viruses, the quantity of genome obtained from clinical samples is very little, so it had to be optimized. First, primer and RNA degeneration were proformed at 65℃for 15min, using a two staged method of reverse transcription, and eliminating the possibility of existence of polymers and dimmers. Then reverse transcription was performed at 37℃for 1h, and the temperature was increased to 85℃for 10min, to eliminate RNA-cDNA hybrids. Second, considering the secondary structure and PolyA tail structure in the template RNA, just using random primers for extensions in the retrovirus process. Although there was a slight decline in specificity, the effect was most stable, and could get more cDNA products. The constructed subclones included 577bp-3581bp sequences of Enterovirus71. It also included viral structural protein sequences VP4, VP2, VP3 and VP1. This had provided the material required for research on Enterovirus grouping and infection, and the development of genetically engineered vaccines.According to the markers used, Real-time PCR detection can be classified into fluorescence probes and fluorescent dyes types. SYBR-Green Q-PCR is a typical representative of fluorescent method. SYBR-Green Q-PCR is a kind of dye which can bind to the small ditch on double-stranded DNA specifically. In the phase of PCR denaturation with high temperatures, the DNA double-strand falls apart and releases no fluorescence. While in the phases of renaturation and extension of PCR, double-stranded DNA is formed and SYBR-Green erupted fluorescence. Then signals are collected at this stage. With more PCR products, more SYBR-Green bands are produced, and more fluorescent signals are collected. Using this method, we could quantify target genes by detecting the release of fluorescence. This method only needs a primer with high specificity instead of expensive synthesized probes. Then we can achieve the purpose of testing by enhancing its specificity.In the second part, we used a quantitative fluorescence polymerase chain reaction (PCR) method to detect hand-foot-and-mouth disease caused by Enterovirus71. Primers aimed at the conservative regions in gene VP1 of Enterovirus71 were designed using the software ABI primer Express2.0. The subcloning which was detailed in part 1 was used as a positive SYBR-Green fluorescence standard curve and quantitative PCR positive controls. Then repeatability tests established an experimental evaluation of the specificity of the methods. We successfully constructed a SYBR-Green fluorescence quantitative polymerase chain reaction (PCR) standard curve:Y=-301x+36.95, with a correlation coefficient of 0.994 and a variation coefficient between the average test of 0.945%.Gene chips can be classified into two categories based on the probes used, namely:Oligonucleotide microarray and cDNA microarray. Oligonucleotide microarrays are made mainly by In-situ synthesis methods, while cDNA arrays are made mainly by microarray chip manufacturing methods. When compared with one another, oligonucleotide microarray has many advantages. While probes on oligonucleotide microarrays are specific DNA oligonucleotide fragments, many genes are hybridized on one chip, while having the same hybridization conditions. However, the length of genes on cDNA chips variy, causing different Tm values and limiting their distinguish-ability. When oligonucleotide microarray is optimized, they can avoid the influence of failed amplification. Therefore, this study used oligonucleotide microarray for testing. With the regard to length of the probe, a 60mer oligonucleotide was chosen in order to get a balance between specificity and sensitivity.In the third part of this study, the subtypes of C4 (registration number:FJ360544) of Enterovirus71 was selected for this experiment. Its genome has an open reading frame, coding a polyphosphate protein containing 2,194 amino acids, and its 5'and 3' ends were non-coding regions. Enterovirus 71 codes 4 structural proteins and 7 nonstructural proteins. BLAST was used to compare the Enterovirus71 full-length genome sequences and the polio virus, Coxsackie virus and Echo virus which are similar to Enterovirus 71. According to the results of the camparison areas of high homology were eliminated, and BLAST was performed a second and third time between these similar segments with allergenic value less than 40 using the software Allele ID 6.0. Finally, the structural proteins VP4, VP2, VP3, and VP1 were chosen as possible alternative sequences for the design of oligonucleotide microarray probes. The Array Designer 4.2 software was used to analyze the specific sequences and 20 probes with uniform and appropriate length were designed. The positive control was marked repeat sequences of general primer and the negative control was rice gene sequences, which have no homology with Enterovirus71. A blank control was also used. Choose the subcloning which has referenced in part one as positive plasmid, and then screened oligonucleotide probes. At the same time RD fluorescence labeled hybridization was performed on Coxsackie's virus samples to test the specificity of the probes. According to a screening standard of an appropriate fluorescent signal value and signal-to-noise ratio,14 oligonucleotide probes with high specificity and high sensitivity were selected. They were:1,2 (according to the VP4 gene), 4,6,7(VP2 gene),9,10,12 (VP3 gene),13,14,16,17,19,20 (VP1 gene). This layed an experimental foundation for the research of multi-virus testing chips.In summary, an alignment analysis was performed through NCBI, a cDNA subclone of Enterovirus71 was built while including the highly conserved regions VP4,VP2,VP3 and VP1. According to the international classification of genes VP1, SYBR-Green fluorescence quantitative polymerase chain reaction (PCR) was performed, and a further quantitative analysis on Enterovirus 71 was done. Then an oligonucleotide microarray of Enterovirus 71 was designed by using biological software, and the 14 high specificity probes that qualified were optimized via experiments, which layed the foundation for testing chips with multiple genes. This research has successfully established a firm foundation for the early diagnosis and therapy of Enterovirus71 infecion.