A Luminex-based Strategy For Multiplex Detection Of Pathogens In Central Nervous System Infectious Diseases

Central nervous system (CNS) infection is a common and multiple disease caused by many pathogens including viruses, bacteria, fungi, parasites and prions, and so on. These diseases usually carry a high rate of mortality and severe sequelae. In order to reduce rate of mortality and improve the cure rate, it is great important to screen and identify pathogens in CSF samples from patients as early as possible. Early and accurate diagnosis will support physicians with the selection of the appropriate antimicrobial agents.Objective Currently, pathogen diagnosis mainly depends on immunofluorescent staining, pathogen culture, and PCR assays. However, traditional methods are susceptible to be interfered by many objective factors. Luminex technology is charactered with high-throughput, accuracy, high speed, sensitivity and specificity, which can qualitatively and quantitatively detect many indicators at the same time. In this study, we developed an early, rapid, high-throughput diagnostic method based on Luminex technology for detecting pathogens. Methods This study covered five important pathogens found in meningitis and encephalitis patients. The pathogens include Tuberculosis, Cryptococcus neoformans, Streptococcus pneumonia, Herpes simplex virus types 1 and 2. We selected the specific genes of five pathogens and obtained conserved gene sequences from the GenBank. These sequences were analyzed by biological softwares and ensure that these sequences are specific. Primers and oligonucleotide probes were designed by oligo6.65. Through bioinformatic analysis and experimental validation we screened out 5 pairs of specific primers and the corresponding probes. The 5' end of oligonucleotide probes were labeled with amino-(CH2)12 modifier and then this modified probes were coupled with microspheres, using as response vector. Through the hybridization reaction between single or multiplex PCR fragments and oligonucleotide probes, we can verify the specificity and sensitivity of detection. Moreover, using these response vectors we detected pathogens in the cerebrospinal fluid (CSF) from the patients with CNS infectious disease and assessed the feasibility of Luminex-based method. Results In the experiments, the results of single nucleic acid amplification showed that specific gene fragments can be achieved. To ensure the consistency and stability of multiplex amplification, we selected 55℃as the optimal annealing temperature. This temperature can be compatible with 5 primer sets in the multiplex amplification. Optimization of hybridization conditions include hybridization temperature and time. Experimental results suggested that optimized hybridization temperature was 55℃, hybridization time was 20min. In this testing system, Mycobacterium tuberculosis, Cryptococcus neoformans, Streptococcus pneumoniae, Herpes simplex virus type 1 and 2 were detected at the same time. PCR validation results showed that five-plex detection system based on Luminex technology was feasible. The sensitivity of the assay was performed with genomic DNA extracting from Mycobacterium tuberculosis, Streptococcus pneumoniae and Herpes simplex virus type 1. The detection sensitivity can attach to fg level. The luminex assay coupled with PCR fragments was able to detect 224 fg/μl for TB, 55 fg/μl for SPN, 35 fg/μl for HSV-1. We also used Luminex technology to detect 41 CSF specimens which were diagnosed as tuberculous meningitis. 39 cases detected were positive. In comparison with golden standard, the compliance rate was 95.1%. Among 17 CSF specimens with negative follow-up results, no one was detected positive. In addition, from suspected tuberculous meningitis CSF samples, 6 cases were detected positive. 20 CSF samples diagnosed as Cryptococcal meningitis were also detected by this system. The results indicated that 19 cases were positive. In comparison with golden standard, the compliance rate was 95%. Conclusion The multiplex detection platform based on Luminex technology is suitable for an initial screening for the important pathogens from patients with central nervous system infection. This rapid, sensitive and high-throught technique can be used as a valuable supplement to the traditional detection methods.