Development On Pathogen Detection Method Of Lower Respiratory Tract Pathogens Based On Multiplex PCR And Next Generation High Throughput Sequencing Technology

As a world-wide public health problem,respiratory tract infection,especially lower respiratory tract infection(LRTI),mainly occurs in people with low immunity such as the elderly and children,which has caused high death rate.Besides,the pathogenic types of LRTI is various,including bacteria,fungi,protozoa,viruses and so on,which substantially rise up the difficulty for clinical rapid diagnosis and accurate medication.There is no doubt that traditional pathogen culture method is the"gold standard",but it has the disadvantages of time consumption and low detection flux.In order to control the disease,it is inevitable to apply empirical drug to the clinic and then adjust it according to the result of culture and drug sensitivity test,which to some extent aggravates the drug resistance to lower respiratory tract pathogens.Therefore,it is of great significance to develop new,more accurate,rapid,and high-throughput detection methods for lower respiratory tract infections.This study combined multiplex PCR and next generation sequencing technology(NGS)to develop a method for detecting lower respiratory pathogenic microorganisms,named AC-seq.AC-seq was preliminarily established and optimized through optimization of breaking conditions for nucleic acid extraction,primers design and validation,and optimization of multiplex PCR detection system;then,thirty-four samples of bronchoalveolar lavage fluid(BALF)from suspected patients with lower respiratory tract infection were identified and compared the results with conventional culture method.First,twenty common pathogens in LRTI were selected as detection targets which includes 12 Gram-negative bacteria,6 Gram-positive bacteria and 2 fungi.Considering the thick cell walls,Enterococcus faecium ATCC 19434 and Candida albicans ATCC 10231 were taken as the representatives to explore the conditions for physical crushing cell walls.Automated grinds with glass beads,zirconia beads and steel beads with different particle sizes and qualities was performed to break cell walls.And it was finally determined that the optimum condition of physical crushing was breaking 0.5 ml bacterial fluid with 0.7 g glass bead mixture of 0.1 mm and 0.5 mm,and the crushing efficiency of two pathogens reached99.97%and 100%,respectively.Meanwhile,40 pairs of specific primers were designed to target 20 pathogens,as each target needed 2 primers.The sensitivity and specificity of primer pairs were confirmed well.Then,Streptococcus pneumoniae ATCC 49619,Acinetobacter baumannii ATCC 19606,Staphylococcus aureus ATCC 43300,Escherichia coli ATCC 35150,Candida albicans ATCC10231 were used as simulate clinical samples for establishing and optimizing the multiplex PCR system.In order to minimize the amount of primer dimer produced by PCR amplification,the ratio of adsorbed magnetic beads,the volume of primer mix,the number of cycles in first round of PCR,the volume of wash buffer and the brand of magnetic beads were optimized or selected.The proportion of adsorbed magnetic beads in first round of PCR was determined to be 0.7×(28μl),the volume of primer mix was 4μl,the number of cycles in first round of PCR was 19,and the ratio of two rounds of wash buffer are 2×BW 15(80μl)and 2×BW 11(80μl),respectively.Combining the above factors,the best magnetic bead is selected from three brands as primer dimer produced by multiplex PCR reaction for simulated samples dropped down to the level that can’t be detected by fluorometer.Besides,the uniformity value of all primers was adjusted from 0～6.57 to 0.1～2.5,which indicated that the amplification efficiency of each pair of primers in the same amplification system was more evenly.And one clinical positive sample was used to compare the results before and after optimization,showing that the content of primer dimer decreased by 55.63%and the sequence reads of target product did not decrease significantly(P=0.44>0.05).The sensitivity of the optimized detection system is confirmed to be 5×10~0copies.Finally,34 clinical bronchoalveolar lavage fluid(BALF)samples were detected with the optimized method and the positive rate reached 79.41%,which was 2.45 times higher than conventional culture(32.35%).Thirteen pathogens were detected from clinical samples by this method,and only eight were detected by culture.Both methods showed that Stenotrophomonas maltophilia and Acinetobacter baumannii were the two main kinds.The concordance rate of two methods was 50%and the sensitivity of optimized method was100%.In conclusion,a detection method of lower respiratory tract pathogens based on multiplex PCR and NGS technology was initially established in this study.It can sensitively and efficiently detect 20 kinds of lower respiratory tract pathogens,which provides certain reference value and application potential for detection of respiratory tract pathogens.