Common Respiratory Viruses With High Sensitivity Rapid Detection Method

Objective:Respiratory tract infection caused by respiratory viruses is one of the main reasons lead to death. In recent years, epidemic outbreaks are also gradually increased, such as avian flu, the H1N1influenza virus, adenovirus as well as the recent outbreak of H7N9influenza virus, which has endangered human’s health toagreatextent. Most of respiratory viral infections are characterized by easy infection, quickly spread, short incubation period, acute onset. The disease and etiology are complicated. Therefore, the rapid and accurate detection and identification of pathogens not only can provide a sufficient evidence for the diagnosis and treatment, also effectively prevent and control the epidemic. It is essential to find a fast and efficient method for respiratory viruses’detection. Currently, the traditional testing methods, such as virus isolation, immunological detection, are not appropriate to promote, for their long cycle period and complicated operation. Although the modern molecular biological technologies such as quantitative PCR, capillary electrophoresis can improve the detection efficiency and shorten the operating cycle period to certain extent, it also unable to simultaneously detect multiple target virus. However, Gene-chip technology has unique advantages in the field of virus detection since it can fast,high-throughput, parallelly detect multiple target gene fragment. The purpose of the subject is to establish a fast, sensitive, specific, high-throughput detection method based on gene chip technology for the eight common respiratory viruses. Meanwhile it also can provide experimental evidence for the identification of respiratory viruses as well as a fast, effective means of diagnosis infections in clinical diagnosis, health surveillance, epidemic prevention and control.Methods:The subject selected eight common respiratory viruses as the target virus to be tested. Based on gene-chip technology, it established highly sensitive and rapid detection method by designing selected primers probe, explore the nucleic acid extraction method, planning multiplex RT-PCR amplification combination and optimizing the hybrid system. In the subject, parameters of nucleic acid extraction, multiplex RT-PCR amplification and hybridization had been optimized by orthogonal experiment and contrast detection method to determine the optimum conditions of the chip and establish the interpretation standard of chip. Through the establishment of standards,we evaluate the method from three aspects of specificity, sensitivity and repeatability,According to the evaluation results of detection method,we infer the detection method whether can accurately detect the eight common respiratory viruses, to evaluate the specificity of the detection method;According to the value of vitro transcription RNA,we evaluate the sensitivity of the detection method;According to the coefficient of variation between the value hybridization signal,we determine the detection method whether be stable and reliable,to evaluate the repeatability of the detection method; Finally,262clinical samples were detected by the detection method of this experiment, which had examined detection methods ability of practical application of the investigated.Results:(1). The common respiratory virus detection method established by the project can screen human metapneumovirus, respiratory syncytial virus, influenza A virus, influenza B virus, parainfluenza virus type Ⅰ, parainfluenza virus typeⅡ, parainfluenza virus typeⅢ, adenovirus. We designed and screened six pairs of specific primers including parainfluenza virus universal primers, and eight specific probes. At the same time, set up internal control probe, to monitor the testing process.(2)We investigate the extraction method of the nucleic acid extracted from respiratory tract viral, choose the method of extraction system of magnetic bead extraction which suitable for use in the automatic detection.(3)The optimum conditions of RT-PCR reaction were determined, reverse transcriptase and hot start polymerase dosage are0.35μ L and1.25μ L, unlabeled primers labeled with biotin primer ratio is1:5, the primer concentration is0.2μM and1μM, concentration ratio of the virus primer in the two RT-PCR reaction tubes are2:2:1:1:1and2:2:1; final annealing temperature is52℃.(4)We optimized the hybridization solution composition and hybrid annealing temperature. The best set of hybridization solution composed is5×SSC,0.8%SDS and2.5%formamide. Hybrid optimal temperature is45℃. Finally, the Cutoff value of respiratory virus probe and the interpretation of standard were determined.(5)Specific evaluation results:the values of positive strain hybridization signal are far greater than the Cutoff values of the corresponding probe, the values of negative strain and blank control hybridization signal values are less than the probe Cutoff value, illustrate the specificity of the established method which can accurately detect the eight common respiratory viruses is very good.(6)Sensitivity evaluation results:this method can detect not less than102CFU/mL vitro transcription RNA, the detection sensitivity than fluorescent chromogenic assay increased100times.(7) Repeatability evaluation results:coefficients of variation between and within are less than15%, indicating that gene chip method for the detection of respiratory viruses is reliable and stable.(8)Detection of262clinical samples shows, the accuracy rate of detection method is96.9%(254/262). Results show that the proposed method can pathogen infection in clinical samples to identify effective, can diagnosis of the respiratory virus mixed infection cases.Conclusion:This study established a method for the detection of gene chip which use quantum dot labeling and silver staining for the detection of innovation of eight kinds of common respiratory virus, with high throughput, fast, good specificity, high sensitivity. And the detection result can be distinguished through visual signal, the operation is convenient and fast. This method can be used for clinical to diagnosis respiratory virus infection of single or mixed infections, and is conducive to the health supervision, control related to the epidemic situation virus outbreak.