Applicatiable Of Luminex And NGS Diagnostic Techniques In The Central Nervous System Infection Pathogens Detection

Background:Central nervous system infections are one of the common causes of central nervous system diseases, with high mortality and disability. There are more than 100 pathogens lead to central nervous system(CNS) infections. Traditional pathogen detection methods(such as microscopy, culture, ELISA test, biochemical, PCR detection, and so on) which have low sensitivity and long diagnostic cycle. These methods can not fultil the requirement of simultaneous identify many kinds of pathogens. Unclear pathogenic diagnosis and empirical antibiotic treatment not only led to the abuse of antibiotics and microbial drug resistance, but also cause a heavy financial burden for patients. Therefore, in order to reduce the mortality and morbidity of CNS infection disease, clinical pathogens for the disease urgently need to be a detection method which could be fast, accurate and high throughput.In this research, we made the Luminex200 liquid chip and next generation sequencing(NGS) technology establish a Two-Level pathogens detection system. for testing CSF samples from unclear pathogenic diagnosis patiens.Part one: base on the Luminex-200 platform a two-tube multiplex PCR assay for simultaneous detection of 18 pathogensObjection: Used Luminex 200-MegPlex array system, a high throughput and high specific multiplex-PCR system(which can simultaneous detect 18 common pathogens in CNS infection disease) was established.Method: First, we established a MultiPlex-Mag-Array–PCR system(MPMA-PCR) system, which include MegPlex array, Multiplex-PCR and Temperatur swtich system three strategies. The MAMP-PCR system can two-tue stimultianeously detect 18 pathogens(Tube1: HSV-1,HSV-2,VZV,CMV,EB,MeV,MuV,JCV,JEV,ECHO,EV71,CA16,HEV;Tube 2:C.neoforman,M.tuberculosis,S.pneumonia,T. gondii,A. cantonensis). The specific gene of 18 pathogens were choosen to synthesis and constructed in 18 plasmids, which were served as control-plasmids. By testing these positive standard contrals, we confirmed that the MPMA-PCR system were established. To evaluate the LODs of MPMA system, a panel of 18 control-plasmids were prepared with dilution from 106 to 102, 50 and 10 copies.Secondly,we collected 177 cerebrospinal(CSF) samples,including:138 defined as possible viral encephalitis CSF samples, 19 pretedsted csf sample, and 20 negtive of any viral infection. All these 177 samples were used to verify the MPMA-PCR system which can tested the clinical samples with high specificity.Results:(1)Establish MPMA-PCR system: By testing standard positive controls, we confirmed that the MPMA-PCR system can two-tube stimultianeously detect specific gene of 18 pathogens. Then all the results were confirmed by sequencing.(2)Testing the clinical sampls: Using MPMA-PCR system test 177 clinical CSF samples, we got pathogen information from 41 samples: 14 samples for HSV-1, 11 samples for HSV-2, 4 samples for EB, ECHO, 2 samples for CMV, VZV and 1 sample for Mev, Ca16, Ev71 and Ev68. All the samples were confirmed by sequencing with 95.48% concordance.Conclusion: we established a MPMA-PCR system based on Luminex-200 platform. This system can two-tube stimutaniously detect 18 pathogens. 23.16%(41/177) samples were positive results, and the specificity were 100%.Part 2 Exploratory application of NGS detect unknown pathogens causing central system infection.Objective: After samples were confirmed by MPMA-PCR, there were 117 samples with no informations of pathogens. For getting more informations of pathogens, We performed to research on application of NGS detection.Method: We collected 33 CSF samples. Among the 33 samples, 21 CSF samples were come from 117 sample with no information of pathogens, by the MPMA-PCR, and 12 CSF samples from the patients who were definitely diagnosed as Cryptococcal meningitis. These 12 CSF samples were serviced as positive control. By NGS technology, we detected 33 samples. qPCR are required for verifying the positive results.Conclusion: By NGS technology, we get 28.57% positive results from the samples with negative PCR results. This results suggested that the NGS technology is feasible in pathogens test with CSF samples. Due to no mature methods of library establishment and bioinformatics analysis, there were many unclear questions should be answered. And we should do more research on that.