Establishment and verification of dual TaqMan probe qPCR detection method for target gene copy number of genetically attenuated pertussis vaccine strains
10.13200/j.cnki.cjb.004490
- VernacularTitle:基因脱毒百日咳疫苗菌株目的基因拷贝数双重TaqMan探针qPCR检测方法的建立及验证
- Author:
BU Xiangli
- Publication Type:Journal Article
- Keywords:
Genetic detoxification;
Pertussis vaccine;
Target gene copy number;
Dual TaqMan probe qPCR;
Passage stability
- From:
Chinese Journal of Biologicals
2025;38(05):595-604
- CountryChina
- Language:Chinese
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Abstract:
Objective To establish a dual TaqMan probe-based qPCR method for determining the copy number of the geneti-cally modified pertussis toxin(PT) gene in detoxified pertussis vaccine strains, and to identify optimal reference genes forevaluating the genetic stability of modified strains during serial passage.MethodsSpecific primers and probes of modifiedgene PT and multiple housekeeping genes were designed. The optimal reference genes were screened by GeNorm, Norm-Finder, BestKeeper and Delta CT based on dual TaqMan probe qPCR. The copy number of PT gene of genetically detoxifiedpertussis vaccine strain was calculated by relative quantitative analysis. The specificity, linearity and repeatability of theestablished method were verified. The genomic DNA of P1, P3, P5, P10 and P15 strains used in the production of geneticallydetoxified pertussis vaccine was used as templates, and the passage stability of the strains was evaluated by using the estab-lished dual TaqMan probe qPCR method.ResultsThe stability of candidate reference genes was analyzed using GeNorm,NormFinder, BestKeeper, and Delta CT software, with Rpob identified as the most stable reference gene. Both the target PTgene and the reference Rpob gene were amplified in standard strain 58003. A good linear correlation(R~2≥ 0. 990) wasobserved between Ct values and template quantities within the concentration range of 0. 001 to 10 ng/μL. For P1 generationstrains, the 2~(-△△Ct)values from six replicate assays consistently ranged between 0. 87 and 1. 25, with an RSD of less than 20%.All tested generations exhibited single-copy PT target genes, demonstrating good genetic stability during serial passages.ConclusionThe established dual TaqMan probe qPCR assay demonstrates good primer specificity and reproducibility,making it suitable for evaluating the genetic stability of bacterial strains during serial passages in the production of geneti-cally detoxified pertussis vaccines, thereby further evaluating the quality of strains.
