Development Of A DNA-based Microarray For Detection Of Nine Pathogens Causing Rash And Fever Illness

Objective: Rash and fever illness(RFIS)take rash and fever(more than 37.5?)as the main clinical manifestations associated with systemic or local skin or mucous membrane rash,and generally may be a series of diseases accompanied with other clinical symptoms.It Includes measles,rubella,typhoid fever,scarlet fever,epidemic hemorrhagic fever,typhus,epidemic meningitis,chickenpox,herpes zoster,smallpox and other infectious diseases,and it is the key monitoring infectious diseases in our country.The main pathogens causing rash and fever illness contains Measles virus,Rubella virus,Enterovirus type 71,Varicella zoster virus,Dengue fever virus,Human small DNA virus B19,Coxsackie virus type A16,A-? hemolytic streptococcus and Salmonella typhi.Most of these pathogens are infectious in the latent period and the clinical symptoms are not obvious.In current,the main detection methods of fever and rash diseases include isolation and cultivation,PCR and immunological diagnosis.None of these methods can meet the needs of detecting a variety of pathogens at the same time.The study is aim to develop a chemiluminescence imaging DNA microarray method for simultaneous,fast and accurate detection of nine rash-and fever-causing pathogens,namely,Measles virus,Rubella virus,Enterovirus type 71,Varicella zoster virus,Dengue fever virus,Human small DNA virus B19,Coxsackie virus type A16,A-? hemolytic streptococcus and Salmonella typhi.Methods: First,the target genes of pathogens and their pathogens were determined by literature investigation.Primers and probes were designed according to the highly conserved regions of the target genes.Second,the clinical samples were collected,and the target genes were spliced and synthesized for the pathogens without the clinical samples,then the plasmid and in vitro transcription RNA reference material were constructed.Third,the nucleic acids of the nine pathogens were amplified and labelled by multiplex PCR method,The multiplex PCR amplification products were hybridized with specific probes of microarray that was scanned after washing and chemiluminescence coloration,and then identify the nine pathogens.After the optimization of the multiplex PCR system,hybridization and chemiluminescence imagination,the specificity,reproducibility and sensitivity of the microarray were evaluated.Fourth,the serial diluted nucleic acid of Enterovirus type 71 was detected using real-time PCR methoed and the microarray to compare the sensitivity of these two methods.Fifth,the practicability of the method was evaluated by the detection of 74 clinical samples.Result: First,according to the survey results of epidemiological data,nine kinds of pathogens were determined.Namely,Measles virus,Rubella virus,Enterovirus type 71,Varicella zoster virus,Dengue fever virus,Human small DNA virus B19,Coxsackie virus type A16,A-? hemolytic streptococcus and Salmonella typhi.Nine specific primers and eleven specific probes were selected.Second,the plasmid reference of these nine pathogens and the in vitro transcription RNA reference of Measles virus,Rubella virus,Coxsackie virus type A16,Enterovirus type 71 and Dengue fever virus were successfully constructed.Third,the optimized multiplex PCR system is divided into A,B two tube,and the microarray performed high sensitivity and specificity.The minimum detection limit of plasmid DNA and vitro transcribed RNAs was 3×103copies per reaction.Fourth,The microarray performed 10 fold lower detection sensitivities than the real-time PCR method.Fifth,the sensitivity rate and specificity rate of 74 clinical samples detection were 95.00% and 85.71%,as well as the accuracy rate 93.24%.Conclusion: A chemiluminescence imaging DNA microarray method for fast,accurate and simultaneous detection of nine pathogens that cause rash and fever illnesses is established successfully,which can serve as a new high throuthput screening method for epidemiological investigation study and clinical diagnosis of rash and fever illnesses.