Application Of GeXP System In Screening Of Congenital Heart Disease Related Gene Mutation And The Multiple Detection Of Respiratory Virus

GeneomeLab Genetic Analysis System(GeXP)was developed by Beckman Coulter Genomics in America,which was mainly used for quantitative analysis of multiple gene expression.The GeXP System is a platform combining multiplex PCR technology and capillary electrophoresis,which could test 35 gene expression abundances from 5ng-500 ng RNA samples in one reaction.GeXP system contains a bunch of eight capillary arrays that make full use of the design arranged in a matrix of 96 orifice plate,can test 192 samples at the same time using the dual channel setting and allow automatic changing of different methods,which is helpful for the quality control and the application of development of method.The precise temperature control and the best resolution are available for the liquid cooling and proper use of semiconductor temperature control in the capillary.In addition,GeXP system equips with three kinds of detector: ultraviolet,diodes matrix and double laser induced fluorescence,and able to cooperate with the different applications.Overall,to a great extent,GeXP system improves the detection sensitivity and speed,and pushes the study of gene expression to a higher level.In recent years,since the GeXP system has the huge advantage;it has been widely used in drug toxicity mechanism research,gene expression regulation of Arabidopsis thaliana associated with iron absorption,the early diagnosis of cancer,immunotherapy,disease classification,individualized medication guide,pathogen identification and drug resistance mutation detection and so on.The present study is divided into two parts: PartⅠ,to simultaneously detect eight mutation loci related with congenital heart disease using the GeXP system,and to analyze the relationship between the eight loci and the onset of CHD;Part Ⅱ,clinical evaluation of a GeXP-based multiplex reverse transcription-PCR assay for simultaneous detection of respiratory viruses,Mycoplasma pneumoniae and Chlamydia pneumoniae.Part Ⅰ Simultaneous detection of eight mutation loci related with Congenital heart disease by a GeXP based multiplex PCR assayObjective: To analyze the relationship between the eight sites and CHD(congenital heart disease)by employing a multiple PCR assay for simultaneous detection of six mutations and two SNPs(single nucleotide polymorphisms)associated with CHD.Methods: Based on GeXP(Genomelab genetic analysis system)and integrated capillary electrophoresis,the specific primers for the detection of eight sites [MTHFR(rs180113 and rs18011331),CRELD1(rs28941780),TBX20(rs137852955),GATA4(rs104894073),TBX5(rs104894381),NKX2-5(rs104893903),TBX1(rs28939675)] of the human genome DNA were designed.The specificity and sensitivity of multiple PCR assay were evaluated,and the optimized assay was further applied to detect 256 CHD samples and 49 healthy controls from the Children’s Hospital of Hebei Province.Chi-square test was used for statistical process with SPSS16.0.Results: A total of 16 genotypes and a control DNA could be amplified in the same reaction tube,and the PCR products were separated distinctively in capillary electrophoresis.The method revealed good sensitivity and specificity.No mutant genotypes of CRELD1,TBX20,GATA4,TBX5,NKX2-5 and TBX1 gene was found in these samples and controls.In contrast,the C677 C genotype of methylenetetrahydrofolate reductase(MTHFR)showed the significant discrepancy between healthy controls and the complex CHD(χ2=4.051,P=0.044,OR=0.211,95%CI 0.041-1.092).Conclusions: The GeXP based multiplex PCR assay provides a simple,reliable and practical method for multi-locus association study of congenital heart disease-related susceptibility genes.Testing of 2 SNPs of MTHFR can be potentially used in prenatal screening and forecast the risk of birth defects.Part Ⅱ Clinical evaluation of a Ge XP-based multiplex reverse transcription-PCR assay for simultaneous detection of respiratory viruses,Mycoplasma pneumoniae and Chlamydia pneumoniaeObjective: To compare the clinical performance of the Respiratory Pathogen 13 Detection Kit(13×-kit)enabling simultaneous detection of 11 respiratory viruses,Mycoplasma pneumoniae(MP)and Chlamydia(Ch)in a single reaction with the GeXP-based multiplex reverse transcription-PCR(RT-PCR)assay(GeXP assay)which has already been evaluated by the commercial RVP Fast assay.Methods: The assay performance was evaluated using 302 Nasopharyngeal aspirates(NPAs)collected from children with acute respiratory tract infections(ARTI)hospitalized in Children’s hospital of Hebei,China from May to October,2015.Nasopharyngeal aspirates(NPAs)were added to 3.5ml of transport medium and stored at-80°C.Results: The sensitivity,specificity,positive predictive value(PPV)and the negative predictive value(NPV)for the 13×-kit were 95.13%,84.21%,94.71% and 85.33%(with GeXP assay as the gold standard),respectively.In a head-to-head comparison for all viruses,the assay concordance was 92.38%,the Kappa coefficient was 0.92.All the MP and Ch detected by 13×-kits were confirmed by Real-time Quantitative PCR(qPCR)or direct sequencing as true positives.Conclusions: The utilization of the 13×kit in clinical testing will be of a great help for clinical doctors to assess the clinical outcome according to virus type or mixed infections,and to limit the use of antibiotics.