Application Of GeXP System Based Multiplex PCR For Screening Aminoglycoside Antibiotics Resistance Genes And Typing Human Enteroviruses Associated With Hand, Food And Mouth Disease

Objective:1. To establish a GeXP based multiplex PCR assay to detect seven aminoglycoside drug resistance genes (aac(3)-Ⅱ, aac(6')-Ⅰb, aac(6')-Ⅱ, ant(3〞)-Ⅰ, aph(3')-Ⅳ, armA and rmtB), and evaluate the application of the sassay for screening the above genes in 56 Klebsiella pneumonia isolates collected from the clinics.2. To develop a GeXP based multiplex RT-PCR assay for simultaneous differentiation of human enterovirus 71 (HEV71), CoxsackievirusA16 (CVA16) and other Coxsackieviruses (CVA4, CVA5, CVA9, CVA10, CVB1, CVB3 and CVB5), and evaluate its application in the detection of a total of 180 clinical specimen.Methods:1. To develop and analyze the GeXP based multiplex PCR assay for detecting seven aminoglycoside drug resistance genes.(1) The sequences of seven aminoglycoside drug resistance genes were downloaded and performed alignment with ClustalX, then specific primers were designed based on the conserved sequences of aac(3)-Ⅱ, aac(6')-Ⅰb, aac(6')-Ⅱ, ant(3〞)-Ⅰ, aph(3')-Ⅳ, armA and rmtB. (2) The genomic DNA was extracted from the bacteria. (3) The specificity of each pair of primers was confirmed via monoplex PCR. A multiplex PCR assay was then established with the combination of proper primers. The products of multiplex PCR were separated and analyzed by GeXP Genetic Analysis System. (4) A 10-fold dilution series of target DNA-containing plasmids with 104copies, 103copies, 102copies and 10copies, respectively, were used to analyze the sensitivity of the GeXP multiplex PCR assay. (5) A total of 56 Klebsiella pneumonia isolates collected from the clinics were screened by the multiplex PCR assay to observe the distributions of aminoglycoside modifying enzymes genes and 16S rRNA methylase genes, and to evaluate the clinical reliability of the GeXP based multiplex PCR assay.2. To develop a GeXP based Multiplex RT-PCR assay for simultaneous differentiation of nine human enteroviruses associated with hand, food and mouth disease.(1) Enterovirus detection was performed with a mixture of 11 pairs of oligonucleotide primers including one pair of primers for amplifying all known pan-enterovirus genomes and ten pairs of primers specific for detection the VP1 genes of EV71, CVA16, CVA4, CVA5, CVA9, CVA10, CVB1, CVB3 and CVB5, respectively. (2) The specificity of multiplex RT-PCR system was examined using cell cultured enteroviruses isolated from hand-foot-and-mouth disease (HFMD) patients. (3) Serial dilution of titrated EV71 and CA16 cell cultures and in vitro transcripted RNAs of enterovirus VP1 genes were used to detect the sensitivity of the multiplex RT-PCR system. (4) A total of 180 unknown specimens were used to evaluate the utility of GeXP multiplex RT-PCR assay in clinical samples. The results were compared with the summary results of traditional typing methods including virus isolation, neutralization test and RT-PCR followed by nucleotide sequencing.Results:1. The detection limit of this multiplex RT-PCR system was 10 copies for 7 aminoglycoside antibiotics resistance genes. In 56 strains of Klebsiella pneumonia, the positive rates of aac(3)-Ⅱ, aac(6')-Ⅰb, ac(6')-Ⅱ, ant(3〞)-Ⅰ, aph(3')-Ⅳ, armA and rmtB were 71.43%, 25.00%, 14.29%, 75.00%, 17.86%, 71.43% and 30.36%, respectively. Compared with traditional monoplex PCR, the sensitivities for different genes were 83.33%～100%, and the specificities were 85.71%～100%.2. The detection limit of this multiplex RT-PCR system was 0.05 TCID50 for titrated HEV71 and CVA16 cell cultures and 100 copies of in vitro transcripted RNAs. The sensitivities of this GeXP based multiplex RT-PCR system for detection of Pan-enterovirus, HEV71 and CVA16 were 98.78%, 91.67% and 91.67%, respectively, and the specificities for detection of Pan-enterovirus, HEV71 and CVA16 were 80.00%, 98.48% and 100%, respectively. The accordance between the detecton results of GeXP based multiplex RT-PCR and the summary results of traditional methods for typing other 7 serotypes enteroviruses was 92.59% (25/27).Conclusion:1. The established GeXP based multiplex PCR assay is demonstrated to be a rapid, sensitive and specific method to screen clinical isolates for the distribution of multi-drug resistance genes of aminoglycosides.2. The established GeXP based multiplex RT-PCR assay is demonstrated to be a rapid, sensitive and specific assay for the diagnosis of common enterovirus infection in cases of HFMD outbreak and is also potentially useful for molecular epidemiological investigations.