Development And Evaluation Of Two Novel Nucleic Acid Test Methods For Detection Of Bordetella Pertussis

Objective:Pertussis is a highly transmissible acute respiratory infection caused by the bacterial pathogen Bordetella pertussis.In recent years,several developed countries with high vaccination coverage have reported that the incidence of pertussis has risen again after maintaining low levels for many years,which considered as a resurgence of pertussis.Therefore,it is significantly important to establish accurate and rapid laboratory diagnostic methods.This research was aimed to establish two nucleic acid detection methods for B.pertussis,which are suitable for various scenes and meet needs of fast and super sensitive detection in different scenes.One is the recombinase aided amplification(RAA)assay incorporating competitive internal amplification control,and the other is the one-tube nested quantitative real-time PCR using the LNA technique.Methods:1. The recombinase aided amplification(RAA)assay incorporating competitive internal amplification control(IAC)to detect Bordetella pertussis using the DNA obtained by boiling:in this study,the forward and reverse primers and probe were designed by based on conserved IS481 gene of B.pertussis,and the IAC probe was derived from previously published article.We established a RAA assay incorporating competitive internal control for diagnosis of B.pertussis,and further evaluated the analytical specificity and sensitivity of internally controlled RAA assay.A total of 115 clinical samples suspected of pertussis were collected and tested by the internally controlled RAA assay using both extracted DNA with the commercial kit and the DNA obtained by boiling.For comparison,the real-time quantitative PCR(q-PCR)based on IS481 gene was also performed with DNA extraction in parallel.2.The one-tube nested quantitative real-time PCR using the LNA technique(LNA-OTN-q-PCR)study targeting BP485 gene:the Inner forward and reverse primers and probe were derived from previously published RT-PCR assay based on conserved BP485 gene of B.pertussis.With modifications by LNA,the outer forward and reverse primers target the outbound region of the inner primers.We established the LNA-OTN-q-PCR assay for diagnosis of B.pertussis,and further evaluated the analytical specificity and sensitivity of LNA-OTN-q-PCR assay.A total of 115 clinical samples from patients with clinically suspected pertussis,collected from the children’s hospital of Hebei,China,were tested by LNA-OTN-q-PCR assay.q-PCR targeting BP485 gene and the combined n PCR and r PCR were also performed in parallel for comparison.Results:1.In conclusion,we performed successfully a RAA assay incorporating competitive internal control for diagnosis of B.pertussis.The sensitivity of the internally controlled RAA assay was 10~1copies per reaction in detecting plasmid DNA.The optimum concentration of the IAC plasmid was determined to be 100 copies,and the introduction of IAC effectively reduced the occurrence of false negatives.Compared to the RT-PCR,RAA results with DNA extraction obtained 100%sensitivity and specificity,and the RAA results with heat-treated DNA showed 85.96%sensitivity and 100%specificity.2. In our study,we performed successfully the LNA-OTN-q-PCR assay for the detection of B. pertussis.Only strains of B.pertussis were identified as positive,whereas all of the remaining strains were appropriately identified as negative by the LNA-OTN-q-PCR assay.A single copy per reaction can be detected by the LNA-OTN-q-PCR assay.Additionally,the sensitivity of this method was 100 times that of the RT-PCR assay(100 copies per reaction).Sixty out of 115 clinical samples were detected positive by LNA-OTN-q-PCR assay,in contrast,RT-PCR was able to detect only 41 positive samples.Following this,all 60 samples had been positively identified by the combined n PCR and r PCR.Compared with the combined n PCR and r PCR assay,both the specificity and sensitivity of the LNA-OTN-q-PCR assay using Purified DNA and Crude extraction were 100%.Conclusion:The two nucleic acid detection methods of B.pertussis established in this study make up for the deficiency of current nucleic acid amplifications,have advantages of rapid and super sensitive in the field,and have high application value in different scenes.It provides reliable gists for etiological diagnosis and epidemiological study of pertussis.It is of great significance for the prevention and treatment of pertussis.