Methodology And Application Of PCR-Membrane Chromatography Hybridization For Six Respiratory Bacterial Pathogens Multiple Detection

Background:Lower respiratory tract infection remains one of the major causes of hospitalization and death worldwide.Acinetobacter baumannii,Escherichia coli,Klebsiella pneumoniae,Haemophilus influenzae,Staphylococcus aureus and Pseudomonas aeruginosa are the main respiratory pathogens causing morbidity and mortality in developing countries.Objective:To establish a rapid,accurate,sensitive,high-throughput,easy to operate PCR-membrane chromatography hybridization assay,which the result could be read by eye and applied for multiplex and semiquantitative analysis of respiratory bacteria To realize the joint detection of 6 respiratory pathogens,including Acinetobacter baumannii,Escherichia coli,Klebsiella pneumoniae,Haemophilus influenzae,Staphylococcus aureus and Pseudomonas aeruginosa.Methods:1.The establishment of PCR-membrane chromatography hybridization assay for multiplex analysis detection of six respiratory bacterial pathogens.For each pair of primers of six bacteria,the 5' terminus of forward primer was tagged with five different oligonucleotides(Tag)and the 5' terminus of reverse primer was tagged with biotin.The terminal of the forward primer was connected with tag by spacer C3.Dipstick strips were manufactured by immobilizing the respective complementary olionucleotides(cTag)in the test area to form 6 test lines for multiplex analysis of target DNA sequences of the lower respiratory tract infection associated bacteria,Acinetobacter baumannii,Escherichia coli,Klebsiella pneumoniae,Haemophilus influenzae,Staphylococcus aureus and Pseudomonas aeruginosa.To ensure the normal function of paper-based microfluidic bioassay,a interal control line was formed at the end of each strip by applying biotin alone.The biotin at the 5'end of the reverse primer combined with the blue latex microspheres which coated with streptavidin to generate signal.Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip.The accumulation of captured latex beads on the test and interal control lines produced blue bands,enabling visual detection with the naked eye.The limit of detection(LOD)and semiquantification of six bacteria was performed by obtaining a series of chromatograms using 10-fold serial dilution of six standard strains PCR-amplified DNA.2.The application of PCR-membrane chromatography hybridization method.The PCR-membrane chromatography hybridization was employed to retrospectively analyze 157 samples of sputum specimens,and the results were compared with the conventional culture method.SPSS 24.0 statistical software was used for data analysis,and paired chi square test was used to calculate the coincidence rate of the PCR-membrane chromatography hybridization assay and conventional culture method.In order to further evaluate the amplification products detection accuracy of PCR-membrane chromatography hybridization,all PCR amplification products were sent to sequencing.Results:1.PCR-membrane chromatography hybridization assay specifically detected and differentiated the amplification products of Acinetobacter baumannii,Escherichia coli,Klebsiella pneumoniae,Haemophilus influenzae,Staphylococcus aureus and Pseudomonas aeruginosa,which showed a high sensitivity and a good specificity.The limit of detection ranged from 10 to 102 CFU/mL of single bacterium using PCR-membrane chromatography hybridization assay.There was no cross reaction among the six bacteria.2.For the detection of 157 samples of clinical sputum specimens,the conventional culture method revealed that there were 24 cases of Acinetobacter baumannii,16 cases of Escherichia coli,23 cases of Klebsiella pneumoniae,5 cases of Haemophilus influenzae,15 cases of Staphylococcus aureus,and 24 cases of Pseudomonas aeruginosa,with semi-quantitative culture being more than ++.Meanwhile,50 cases were negative samples.The 11 samples out of 107 culture positive were identified as 2-3 bacterial co-infections and and 96 cases as were identified as single pathogen infection by PCR-membrane chromatography hybridization assay.Meanwhile,and with 50 culture negative samples were confirmed as negative by PCR-membrane chromatography hybridization assay.The consistency between PCR-membrane chromatography hybridization and DNA sequencing was 100%,and kappa value was 1.00.Compared with the bacterial culture which was regarded as the gold standard of clinical diagnosis,the positive predictive value of PCR-membrane chromatography hybridization assay was 89.7%and the negative predictive value was 100%.In general,for the detection of 157 clinical samples,the consistency of PCR-membrane chromatography hybridization and bacterial culture was 93.0%.The coincidence rates of Acinetobacter baumannii,Escherichia coli,Klebsiella pneumoniae,Haemophilus influenzae,Staphylococcus aureus and Pseudomonas aeruginosa were 95.8%,75.0%,91.3%,80%,93.3%and 91.7%,respectively.Conclusions:A PCR-membrane chromatography hybridization method was successfully established for the joint detection of six respiratory tract bacterial pathogens,including Acinetobacter baumannii,Escherichia coli,Klebsiella pneumoniae,Haemophilus influenzae,Staphylococcus aureus and Pseudomonas aeruginosa.It is simple,rapid,high throughput,sensitive,accurate,cost-effective and has a good diagnostic performance,which can be used for small laboratories and point-of-care diagnosis.