Development Of Detection Methods Of Micro-chips For Three Respiratory Tract Viruses

Acute respiratory tract infection (ARTI) is the most common infectious diseasesof human, which is also one of the main factors endangering the health of children.Clinical data showed that most ARTI were caused by respiratory viruses.Rhinovirus,respiratory syncytial virus and adenovirus are the common pathogens causing ARTI.The rhinovirus (Rhv) belonging to Picornaviridae is the major pathogen thatcauses the common cold. RSV is a member of the genus Pneumovirus in the familyParamyxoviridae and it has a negative-sense, single-stranded RNA genome. RSV is amajor cause of ARTI among infants and young children. In china, RSV is associatedwith the disease Enzootic pneumonia. The adenovirus is a double-stranded DNA virus,which can exist in the human body for a long time and may account for the large-scaleepidemic of ARTI.Currently there are many methods for detecting the respiratory tract viruses, theregular ones include virus tissue culture isolation test, direct immunofluorescent assay(DFA), indirect immunofluorescent assay (IFA), enzyme-linked immunosorbent assay(ELISA), multiplex real-time PCR, nested PCR, nucleic acid hybridization,suspension array technology, and so on. Although these classical methods have theirown features, they also have the shortcomings, such as, consuming time of thedetection process, higher cost, low flux, weak specificity and sensitivity. Therefore, itis necessary to establish a rapid method for detecting Rhv, RSV and Adv. Because ofthe inherent features of high-throughput, microscale, and convenient, microarraytechnology has the superior advantages over traditional detection technologies, thus,this approach has been widely used for effectively detecting the respiratory tractviruses. In attempt to establish a method for rapidly detecting Rhv, RSV and Adv, a genechip technology was developed in the present study and preliminary verification andevaluation was also proceeded.First of all, gene sequences of three viruses were screened from the GenBank andsubsequently were multiple-aligned by using the program MegAlign in DNASTARpackage. According to the multiple alignment results, specific primers and probestargeting the conservative regions of the respective viruses were designed throughusing the biological software Primer5.0and Array Designer4.20. To ensure thespecificity of the detection, two or three probes for each virus were designed.The second part is about the preparation of gene microarray and the preliminaryverification. After amplification by nested PCR, the target gene fragments of threeviruses were labeled with fluorescent substance. The labeled nucleic acid fragmentswere then individually hybridized with the probes fixed on the optical grade aldehydeslides and the chips were washed to remove unbound gene fragments, which werethen subjected to the chip scanner for scanning the hybridization results. On the basisof the correct preliminary verification, the conditions including Liquid formula,hybrid liquid formamide concentration, hybrid time and temperature were optimized:It is the best hybrid effect to select Liquid formula made myself, hybrid liquidcontaining of10%formamide,42℃and3-6h.In the third part of this study, the performance of the gene chip was evaluatedwith specificity test, sensitivity test,reproducibility test and preservation test.For thespecificity test, recombinant plasmids containing parainfluenza and coxsackievirusgenes and cDNA of Avian influenza virus and Newcastle disease virus prepared in ourlaboratory were selected as control, and the specificity of the chip was verified. Forthe sensitivity test, positive plasmids of Rhv, RSV and Adv were serially diluted toevaluate the sensitivity of the established chips. These positive plasmids were alsoused as the template for the reproducibility test, in which five repeats for intra-chipsand inter-chips were performed under the same experimental conditions. To determine the best storage time and temperature of the chip, the experiments were performed ina200-day preservation test.Microchips for detecting three respiratory tract viruses Rhv, RSV, and Adv weresuccessfully established in the present study. These developed methods have highspecificity and reproducibility; The detectable copy numbers of the chips for Rhv,RSV and Adv were2.59×102copies/L,2.21×101copies/L and2.05×102copies/L.The sensitivity of these chips was10times higher than that of the RT-nPCR methodpreviously established in our experiment; The validity period of prepared chip is about160days at-20°C.Therefore these methods can be used to detect clinical suspectedrespiratory tract infection samples.