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- Title
猫杯状病毒RAA-CRISPR/Cas12a-LFS检测方法的建立及初步应用.
- Authors
周红蕾; 程淑琴; 李佳鹏; 刘涵; 卓国荣; 张蕾
- Abstract
To establish a method for rapid detection of feline calicivirus (FCV) based on lateral flow chromatography strip (LFS), recombinant enzyme-mediated isothermal nucleic acid amplification (RAA) combined with CRISPR-Casl2a system (RAA-CRISPR/Casl2a-LFS), this study designed RAA primers, four amplification primers targeting VPl, four VPl-crRNA amplification primers, and one reporter probe based on the conserved region of the FCV capsid protein (VPl) gene. Four pairs of VPl-crRNA amplification primers were annealed and transcribed by T7 RNA polymerase to synthesize four target crRNAs: VPl-l-crRNA-VPl-4-crRNA; four pairs of VPl target amplification primers were annealed to synthesize four target double- stranded DNA: VPl-l-VPl-4, after the cleavage reaction of EnGen® Lba Casl2a (Cpfl) nuclease reagent and the detection of Casl2/13 strip, the best VPl-crRNA was screened according to the intensity of the bands. The VPl gene of FCV RNA isolated from FCV VR-782 strains and the other six strains was amplified by RAA primer, and the amplification effect ofRAA primer was detected and evaluated by agar-gel electrophoresis. Using Casl2a (Cpfl) nuclease, the VPl gene (from FCV VR-782 strain RNA) amplified by RAA was used as the template, and the product was subjected to a constant temperature water bath at 37°C for 30min for CRISPR/Casl2A-LFS detection. To evaluate the importance of RAA products, Casl2a, VPl-2-crRNA, and ssDNA reporter groups for CRISPR/Casl2A-LFS assay systems. The screening results ofVPl-crRNA showed that the T-line band corresponding to VPl-2-crRNA was the strongest and the band with incomplete C-line cutting was the weakest. The results of RAA amplification showed that the VPl gene target band of 282bp could be amplified from FCV VR-782 strains and FCV RNA isolated from six strains. The importance evaluation results showed that the CRISPR/Casl2a-LFS assay system could be amplified effectively when the four above reagents were present. FCV, feline herpesvirus type I, bordetella bronchosepticus, feline parvovirus, Salmonella, Staphylococcus aureus and F8 l cells were detected by this method. The results showed that the method could detect FCV specifically, while the detection results of the other pathogens were negative. RAA amplification of FCV was completed within 30min at 42°C, and CRISPR/Casl2a-LFS detection of FCV was completed within 40min at 37°C . The method's sensitivity was evaluated using recombinant plasmid standard pMD18T-FCV-VPl with 10-fold dilution. The detection limit of this method was 37.5 copies/μL, comparable to the sensitivity of the TaqMan fluorescence quantitative PCR method established in our laboratory. The same batch and three batches of RAA-CRISPR/Casl2a-LFS system were used to detect recombinant plasmid standard pMD18T-FCV-VPl with different concentrations, respectively, to evaluate the method's reproducibility. The results showed that the method's reproducibility was consistent within and between batches. The method and TaqMan fluorescence quantitative PCR method were used to detect 76 cat respiratory tract swab samples. The results showed that 37 FCV-positive and 39 FCV-negative samples were detected. TaqMan fluorescence quantitative PCR detected 36 FCV-positive samples and 40 FCV-negative samples. The positive coincidence rate was 97.30%, the negative coincidence rate was 97.50%, and the total coincidence rate was 98.68%. The FCV RAA-CRISPR/Casl2a-LFS detection method established in this study is simple, rapid, and has strong specificity, high sensitivity, and good repeatability. This method does not rely on expensive equipment, providing a new technical means for detecting FCV in the field.
- Subjects
NUCLEIC acids; RNA polymerases; GENE targeting; DETECTION limit; STAPHYLOCOCCUS aureus; POLYMERASES; PARVOVIRUS B19; PARVOVIRUSES
- Publication
Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao, 2023, Vol 45, Issue 5, p494
- ISSN
1008-0589
- Publication type
Article
- DOI
10.3969/j.issn.1008-0589.202208013