[Study on the internal control on polymerase chain reaction in Yersinia pestis detection].
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.


CONCLUSIONS
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.
Zhang
Zhi-kai
ZK
Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Hai
Rong
R
Zhang
En-min
EM
Yu
Dong-zheng
DZ
D001426:Bacterial Proteins / Q000032:analysis*; D004269:DNA, Bacterial / Q000032:analysis; D005188:False Negative Reactions; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S; D012680:Sensitivity and Specificity; D015010:Yersinia pestis / Q000302:isolation & purification*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; C086725:caf1 protein, Yersinia pestis