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Updated: Aug 17, 2026
Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis
Published on: June 17, 2019
[Study on the internal control on polymerase chain reaction in Yersinia pestis detection]
Zhi-kai Zhang1, Rong Hai, En-min Zhang
1Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Objective:
For the detection of Yersinia pestis by polymerase chain reaction (PCR), internal control (IC) is required in order to prevent false negative results that might be caused by PCR inhibitors.
Methods:
F1 antigen was amplified by PCR with primer F1 and the PCR product of primer F1 were cloned with TOPO TA cloning Kit. The plasmid of positive clone was then digested with HpaI. The digested plasmid and the PCR products of 16S rRNA were ligated with T(4) DNA ligase before the ligated products were transformed. Isolate plasmid DNA on positive clone and its concentration were measured. Plasmid DNA on different concentration by PCR amplification with primer F1 was analyzed and the standard concentration of IC was determined.
Results:
Constructing an IC by inserting a 16S rRNA amplicon to the original target DNA between the two primer F1 sites, the size was longer than the target DNA. The standard concentration of IC was determined.
Conclusion:
An optimal IC concentration to increase the reliability of the PCR assays might be used to prevent false negative results and appeared to be useful for detection of Yersinia pestis.
Insights
An internal control (IC) was developed to enhance the reliability of polymerase chain reaction (PCR) assays for detecting Yersinia pestis. This optimized IC concentration effectively prevents false-negative results caused by PCR inhibitors.
Area of Science:
- Molecular Biology
- Microbiology
Background:
- Polymerase chain reaction (PCR) is crucial for detecting Yersinia pestis.
- False-negative results in PCR can occur due to PCR inhibitors.
- Internal controls (IC) are necessary to validate PCR assay results.
Purpose of the Study:
- To develop and optimize an internal control (IC) for Yersinia pestis detection using PCR.
- To ensure the reliability of PCR assays by preventing false-negative outcomes.
Main Methods:
- F1 antigen was amplified via PCR, and the product was cloned.
- A 16S rRNA amplicon was inserted into the target DNA to create the IC.
- The optimal concentration of the IC was determined through PCR analysis.
Main Results:
- An IC was successfully constructed by inserting a 16S rRNA amplicon, resulting in a larger product size than the target DNA.
- The standard concentration for the IC was determined.
Conclusions:
- The developed IC can be used at an optimal concentration to improve PCR assay reliability.
- This method effectively prevents false-negative results in Yersinia pestis detection.
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