Establishment and application of a duplex TaqMan fluorescence quatitative PCR assay for Mycoplasma gallisepticum and Mycoplasma synovialis detection
10.16303/j.cnki.1005-4545.2025.05.14
- VernacularTitle:鸡毒支原体与鸡滑液囊支原体双重TaqMan荧光定量PCR方法的建立与应用
- Author:
Zhimin DONG
1
;
Lili WANG
1
;
Xiangxue TIAN
1
;
Chao LU
1
;
Li ZHANG
1
;
Minghua YAN
1
Author Information
1. 天津市农业科学院畜牧兽医研究所,天津 300381;天津市畜禽分子育种与生物技术重点实验室,天津 300381;天津市畜禽健康养殖工程技术中心,天津 300381;国家动物疫病天津观测实验点,天津 300381
- Publication Type:Journal Article
- Keywords:
Mycoplasma gallisepticum;
Mycoplasma synoviae;
real-time PCR;
differential diagnosis
- From:
Chinese Journal of Veterinary Science
2025;45(5):987-993,1025
- CountryChina
- Language:Chinese
-
Abstract:
To rapidly detect and differentiate Mycoplasma gallisepticum(MG)and Mycoplasma synovialis(MS),two sets of specific primers and TaqMan probes were designed in this study based on the conserved regions of the 16S rRNA gene of two pathogens in NCBI.A dual TaqMan fluorescence quantitative PCR method for simultaneous detection of MG and MS was established by optimizing the reaction conditions,and the specificity,sensitivity,repeatability,and reliability of the method were verified.The results showed that this method could specifically amplify MG and MS without cross reactivity with 21 pathogens.The sensitivity experiment results showed that the detection limits of this method for MG and MS were 5.40×10 1 copies/μL and 6.60 × 10 1 copies/μL,and the sensitivity was 10 to 100 times higher than that of known methods.In addition,the re-sults of repetitive experiments showed that the coefficient of variation within and between groups was less than 1％.Compared with the single PCR amplification method in NY/T 553-2015,the positive sample detection coincidence rate,negative sample detection coincidence rate,and total sample detection coincidence rate were all 100.00％,indicating the strong reliability of this method.Using this method to detect 84 suspected Mycoplasma infected chicken samples,the results showed that the MG positivity rate was 32.14％(27/84),the MS positivity rate was 22.62％(19/84),and the positivity rate of samples infected with MG and MS was 16.67％(14/84).Concurrent-ly,182 healthy chicken cloacal swab samples,118 healthy chicken nasal swab samples,and 74 chicken farm environmental samples were detected,and the results showed that all samples were positive for Mycoplasma.The above results indicate that this method can be applied to the detec-tion of various clinical samples.In summary,the method established in this study had the advanta-ges of high specificity,high sensitivity,and good reproducibility.It could be used for clinical differ-ential diagnosis,epidemiological investigation,and pathogen purification of MG and MS infections.
