Development Of Rapid Nucleic Acid Detection Methods For Brucella

Brucellosis is a zoonotic infectious disease caused by intracellular Brucella,which seriously affects the development of animal husbandry in China and causes great economic losses.At present,the detection of brucellosis in China mainly base on bacteriological and serological detection methods.However,these methods have the disadvantages of time-consuming and laborious,false positive,and inability to distinguish the death and viable states of Brucella.At the same time,the pathogen can not be detected quickly and the species can not be identified neither.Therefore,it is of great significance to develop rapid detection methods of brucellosis for the prevention and elimination of brucellosis.This project is devoted to the research of rapid detection methods of brucellosis.Based on the nucleic acid amplification technology,a series of detection methods were established to provide practical and effective technical means for the scientific research,prevention and elimination of brucellosis.1.A rapid PMA-qPCR detection method for counting the numeber of viable Brucella was established by combining the PMA with real-time quantitative PCR?qPCR?.The standard recombinant plasmid inserted with the single-copy BCSP31gene fragment was constructed for standard curve building.The qPCR reaction condition,application concentration and exposure time of PMA were all optimized.The specificity,sensitivity,repeatability and other aspects of the PMA-qPCR detection method were analyzed.The results showed that there was a good linear relationship between the CT value and the logarithmic concentration of the copy number of the standard plasmid.The sensitivity was 10³ CFU/mL,which was 100times more sensitive than ordinary PCR methods and the specificity and repeatability were good;Quantitative analysis of the live/dead Brucella mixed sample confirmed that the measured value of the viable count was similar to the theoretical value,and it was consistent with the change rule of the viable count.The number of intracellular viable bacteria after B.Suis S2 infection of macrophages was determined together with plate counting method.There is a statistical difference between the measured values of the number of Brucella,but the order of magnitude are the same,Which may be related to the entry of some intracellular Brucella into the non-culturable state in vitro.This method is suitable for rapid evaluation of the number of viable Brucella,and has certain applicability in studying the parasitic capacity and infection status of Brucella in vivo or intracellularly.2.A rapid visual detection method for Brucella was established based on Recombinase Polymerase Amplification?RPA?technology.Several pairs of primers were designed for bcsp31 gene and screened.When SYBR Green I was introduced for visual detection of RPA amplification products,in order to eliminate the background signal of RPA,the primer concentration and amplification time were optimized.Then the specificity,sensitivity and repeatability of the method were evaluated,and three simulated samples of serum,beef and milk were detected by the method.The results showed that when using the optimal primers for visual detection using RPA technology,after 15 minutes of amplification at 40°C,2?L of 50×SYBR Green I was added and visualization can be achieved with UV torch at 395 nm.The sensitivity of this method is 1.41358×10^-3 ng/?L,the specificity and repeatability are good.The detection results of the simulated samples by this method are consistent with the detection results of Bruce-Ladder test of"GB/T 18646-2018 Diagnostic Techniques for Animal Brucellosis"and this method does not require complicated and expensive instruments,and the detection time is short.It provides an applicable method for the field detection of Brucella in areas with relatively backward detection conditions.3.RPA primers were designed for the genomic difference sequence of B.abortus,B.melitensis and B.suis.Multiple RPA systems were constructed,and the reaction conditions were optimized to establish a multiple RPA detection method for simultaneously detecting B.abortus,B.melitensis and B.suis.Then the specificity,sensitivity and repeatability of the method were evaluated,and three simulated samples of serum,beef and milk were detected by the method.The results showed that the multiplex RPA detection method established in this study was performed on samples by specific primer sets.The sensitivity was 10 pg/?L for B.abortus,1 pg/?L for B.melitensis and B.suis;The detection results of the simulated samples by this method are consistent with the detection results of Bruce-Ladder test of"GB/T18646-2018 Diagnostic Techniques for Animal Brucellosis".This study established a multiple RPA detection method that is fast,sensitive,and specific,and can simultaneously detect B.abortus,B.melitensis and B.suis.Brucella outbreaks caused by B.abortus,B.melitensis and B.suis are as high as 93%.The established multiple RPA detection method for B.abortus,B.melitensis and B.suis can be used for the identification of most isolates of brucella.4.Developed a Brucella visual PCR detection kit based on Bruce-Ladder method,the national standard for diagnostic techniques for animal brucellosis.The kit improves the Bruce-Ladder detection method,and can perform on-site species identification and detection of Brucella,while simplifying the detection conditions and steps,and greatly shortening the detection time.This kit contains PCR Mix,upstream and downstream primers,and SYBR Green I mixture in the eight-tube tube,and it also includes ddH₂O and positive quality control template.As for testing,adding the mix the sample with ddH₂O into the eight-tube tube for PCR amplification procedure is needed only.After the reaction is completed,each tube is irradiated with an UV torch of 300 nm to 500 nm.According to the color or fluorescence brightness of the reaction system at the bottom of each tube,the identification of Brucella species can be completed.This kit has the advantages of strong specificity,high sensitivity,good reproducibility,simple and fast operation,easy to determine results,and convenient field application.It provides tools for Brucella species identification and field detection applications.In summary,three methods for detecting Brucella were established,and a Bruce-Ladder detection kit was developed,which can not only realize the rapid quantitative detection of the number of viable Brucella,but also complete the rapid visual detection of Brucella and the identification between species.This work provides a technical support for the prevention and control of brucellosis.