Detection Method Of Nocardia Based On SS-PCR And RAA Technology

Background:Nocardia is a kind of aerobic actinomycetes with gram positive staining and weak antacid positive,widely distributed in the natural environment.As the infection rate of Nocardia increases year by year,people gradually pay more attention to this pathogen.At present,N.farcinica and N.brasiliensis are common pathogenic strains.Due to the slow growth of Nocardia,low clinical attention and confusing morphological characteristics,the traditional identification methods are easy to miss or misreport,resulting in serious adverse consequences.The housekeeping genes commonly used in the detection of Nocardia reported now are highly conserved,with little difference between species,and can be accurately confirmed by sequencing.Based on the use of specific genes,this study helps to improve the detection of Nocardia to the species level and establish a more rapid and accurate detection method,which is not only vital for animal disease control and prevention,treatment and public health security,but also plays an important role in the immediate initiation of control measures in the grass-roots area,providing a theoretical basis for the detection,treatment and prevention and control measures of Nocardia.Purpose:1.Specific genes were screened by the previous research group,specific primers were designed for this segment,and a method was established for the detection of N.farcinica and N.brasiliensis by Species-specific Polymerase chain reaction(SS-PCR).2.Combines isothermal amplification technology with probe with higher sensitivity,establishing Real-time Recombinase-aided amplification(RT-RAA)method for detection of N.farcinica.3.The Lateral flow analysis strip combined with recombinant-aided isothermal amplification technique(RAA-LFD)for detection of N.farcinica is established.Method:1.Specific primers were designed based on the species-specific genes of N.farcinica and N.brasiliensis NFA＿41530 and O3I＿RS18895 screened by the previous research group,and rapid and accurate dual SS-PCR was established by optimizing reaction conditions and systems.95 strains of N.farcinica,13 strains of N.brasiliensis and 42 non-target strains were amplified to analyze the specificity of the method,and the sensitivity of the SS-PCR method was evaluated by using plasmid and standard strains.Finally,the accuracy of the method was verified by simulated sputum samples,and its value in clinical diagnosis was estimated.2.Specific labeled primers and probes were designed using the conserved region of specific gene NFA＿41530,and the RT-RAA method was established to detect N.farcinica by optimizing the reaction time and temperature.We identified 86 strains of N.farcinica and 46 strains of related isolates,and analyzed the specificity of the method.Then,plasmid and standard strains were used to evaluate its sensitivity.Meanwhile,the sensitivity of the method was compared with that of Real-time Quantitative PCR(q PCR).Finally,the clinical diagnosis effect of this method was evaluated with simulated sputum samples.3.The primers and probes were designed based on recombinant-aided isothermal amplification technology,and the amplified products were detected by combining with flow chromatography test paper technology,so as to establish a RAA-LFD method for the detection of N.farcinica.In addition,45 strains of N.farcinica and 28 related strains were detected to evaluate the specificity of the method.The sensitivity of the method was verified by gradient dilution of the recombinant plasmid.Finally,sputum simulation samples were used to evaluate the detection ability of the method.Results:1.A clear and single target band was amplified for N.farcinica and N.brasiliensis,respectively,while the other 42 non-target strains were not amplified,indicating that the SS-PCR method for detection of N.farcinica and N.brasiliensis has good specificity.The detection limits of the method were 1.3×10~4 copies/μL and 2.4×10~4 copies/μL,respectively,with high sensitivity.The simulated sputum samples of 20 healthy people were used,and the detection coincidence rate was 100%.2.The RT-RAA method for the detection of N.farcinica showed no cross reaction,and the specificity was 100%.The lower limit of RT-RAA detection of Nocardia genomic DNA per reaction was 10 copies/μL,which was 10 times more sensitive than conventional q PCR.The coincidence rate of sputum simulation samples was 100%.3.The RAA-LFD established in this study was used to detect N.farcinica,among which 45 strains of N.farcinica showed two bands,and 28 related strains showed color bands in the quality control line,but no color bands in the detection line,with a specificity of 100%.The lower limit of detection for Nocardia mela was 10~2 copies/μL.The coincidence rate of sputum simulation samples was 100%.Conclusion:1.The SS-PCR method established is the first time to detect N.farcinica and N.brasiliensis by using the full length of specific genes as the target fragment of amplification,which not only strictly guarantees the specificity of the detection method,but also reduces the cost of large-scale screening.2.The RT-RAA method established for the detection of N.farcinica is free from the constraints of complex real-time quantitative PCR instruments,and does not require cyclic amplification at variable temperature,which greatly reduces the reaction time.3.The detection of N.farcinica by RAA-LFD established makes up for the shortcomings of existing techniques,and the visual results of direct observation strips are very suitable for rapid field detection.