Abstract:

Objective: A dual-target real-time fluorescent PCR technique was developed for the detection of IS6110 and IS1081 genes of Mycobacterium tuberculosis complex (MTBC), to evaluate the feasibility of MTBC rapid detection in cow samples, and to provide basic data support for the application of molecular detection technology in the early diagnosis of bovine tuberculosis. Methods: MTBC real-time fluorescence PCR system was established with IS6110 and IS1081 as specific target genes. The limit of detection for two-target real-time fluorescent PCR system was determined by preparing and detecting bovine lung tissues containing different concentrations of Mycobacterium bovis suspensions. Using DNA of 21 common standard nontuberculous mycobacteria (NTM) strains as amplification template, the specificity of dual-target real-time fluorescent PCR detection of MTBC was determined. MGIT 960 liquid culture, Xpert MTB/RIF (Xpert), Xpert MTB/RIF Ultra (Xpert Ultra) and dual-target real-time fluorescent PCR were used to detect the tissue samples. Using liquid culture results as a reference standard, the efficacy of dual target real-time fluorescence PCR detection in detecting MTBC in dissected cow diseased materials was analyzed. Results: According to the results of dual-target real-time fluorescent PCR detecting the DNA of 21 NTM strains, no amplification curve was detected, the detection specificity of MTBC was 100.0% (21/21). The positive rates of liquid culture, Xpert, Xpert Ultra, and dual-target real-time fluorescent PCR detection were 64.0% (32/50), 56.0% (28/50), 78.0% (39/50), and 76.0% (38/50), respectively. The positive rate of dual-target real-time fluorescent PCR detection was significantly higher than that of Xpert detection (χ²=4.456, P=0.035). There was no statistically significant difference between the positive rates of dual-target real-time fluorescent PCR detection and Xpert Ultra detection (χ²=0.056, P=0.812). Based on the results of liquid culture experiments, the sensitivity and specificity of dual-target real-time fluorescent PCR for detecting MTBC were 90.3% (28/31) and 40.0% (6/15), and those of Xpert Ultra were 96.8% (30/31) and 40.0% (6/15), respectively. Conclusion: The dual-target real-time fluorescent PCR technique can be used for early diagnosis of bovine tuberculosis, which is helpful to control the epidemic and spread of zoonotic tuberculosis.

Key words: Tuberculosis, bovine, Animal diseases, Molecular probe techniques, Diagnosis