Multiple Pcr/meningitis, And Encephalitis Haemorrhagic Fever Pathogens - Mass Array Detection Technology

Purpose:The progress of human civilization did not prevent the spread of infectious diseases. Infectious disease prevention is still one of the challenges which human must be faced in the twenty-first Century. Therefore, the study of infectious pathogens is always a task of great significance. In recent years, along with biological technology progressing, the detection and identification technologies for infectious pathogen develop rapidly. All of these new technologies benefits human beings with better life standards. Currently, many molecular techniques are successfully applied on the detection of pathogens, such as PCR technology, gene chip technology, molecular hybridization technique and so on. Compared with the traditional testing technology, the new generation of molecular biological technology is simple operation, time saving, and high detection efficiency, but there are also corresponding disadvantages. How to avoid these disadvantages and develop new technologies has become the primary task of scientific research workers. Based on a multiplex PCR-Mass ARRAY technology which initially was used for genotyping, mutation detection, resistance loci analysis, we successfully developed a multiple PCR-Mass ARRAY platform on the detection of pathogens of infectious diseases. The aim of this study was to establish a multiplex PCR-Mass ARRAY system to detect15pathogens which can cause encephalitis/meningitis and16viruses cause viral hemorrhagic fever. The technology can provide a high throughput, high accuracy and high sensitivity detection method for epidemiological studies as well as screening and identification of infectious pathogens.Methods:(1) Primers design: First, the complete genome sequences of each pathogen were downloaded from Genbank and the conserved regions of these sequences were selected for primers designing by DNA sequence alignment and bioinformatics analysis. For the multiple PCR-Mass ARRAY platform, a pair of amplification primer and one extended primer were needed for each pathogen.(2) Optimilization of primers:The specificity and reproducibility of the primers were optimilized and validated by ddH2O, normal human genomic DNA and artificial recombinant plasmids. The sensitivity of primers was tested by the serially diluted plasmids. After optimilization and establishment of the Multiplex PCR-Mass ARRAY platform for diagnosis of encephalitis/meningitis and viral hemorrhagic fevers,6cultured encephalitis viruses and14clinical suspected encephalitis specimens were used to evaluate the accuracy and sensitivity of the platform. At the same time, RT-PCR and Sanger sequencing were performed in parallel to compare the specificity and sensitivity of the multiple PCR-Mass ARRAY technology with the the above methods.Results and Conclusion:(1) The Multiple PCR-Mass ARRAY detection technique of encephalitis/meningitis pathogens we established is highly specific. The detection system did not produce nonspecific reaction and could not cause false positive results. To determine each plasmid’s detection sensitivity, we used the Multiple PCR-Mass ARRAY to test the serially diluted recombinant plasmids, and found that the sensitivity of encephalitis/meningitis plasmids ranged from100-10000copies/μl, and only two plasmids were10000copies/μl. The test results of6cultured encephalitis viruses showed that all of the viral cDNAs could be detected, with no false positive results. We also used the Sanger sequencing method to test the same samples in parallel, found that the results of the two methods are in consistent. The test results of14clinical suspected encephalitis samples by Multiple PCR Mass ARRAY showed that3samples were positive for Encephalitis B virus (JEV), no viruses were detected in the remaining samples. The results of RT-PCR using the same batch of samples showed two samples were JEV infected, and the two positive samples of real time PCR were among the3JEV positive samples of Multiple PCR-Mass ARRAY. Sanger sequencing results were the same as Multiple PCR-Mass ARRAY. The results showed that the sensitivity of Multiple PCR-Mass ARRAY technology was higher than RT-PCR technique.(2) The established PCR-Mass ARRAY for detecting of16hemorrhagic fever viruses also had good specificity and reproducibility; Serially diluted recombinant plasmids were used to evaluate the sensitivity of the platform, and found that the sensitivity of hemorrhagic fever plasmids ranged from10-100copies/μl, most of them were100copies/μl. The results showed that Multiple PCR-Mass ARRAY technology for detecting of hemorrhagic fever viruses also had higher sensitivity.The results of this study showed that Multiple PCR-Mass ARRAY platform for detecting of encephalitis/meningitis pathogens and hemorrhagic fever viruses has good specificity and reproducibility, and has a relatively higher sensitivity and accuracy compared with real time PCR. Therefore, this technique could be used for epidemiological surveillance and large-scale screening of infectious pathogens.