[The use of internal controls of different lengths in the detection of Chlamydia trachomatis DNA by the polymerase chain reaction method].
To detect C. trachomatis DNA, the polymerase chain reaction (PCR) with the use of primers corresponding to variable sites of rRNA gene 16S was carried out. As the positive control of the reaction, the amplification fragment of gene 16S of rRNA, cloned in the plasmid vector and having the length of 530 nucleotide pairs (n.p.), was used. On its basis 2 kinds of the internal control of the reaction were obtained with the deletion of 110 n.p. (pMOS-Chl420) and the insertion of 930 n.p. (pMOS-Chl1460) within the cloned amplification fragment. The study revealed that the addition of the DNA of pMOS-Chl420 or pMOS-Chl1460 into the reaction mixture did not affect the sensitivity of PCR (0.02 pg of bacterial DNA in the sample) in the detection of C. trachomatis DNA isolated both from the culture of bacterial cells and from clinical samples. But in some cases of the amplification of the DNA of internal control pMOS-Chl420, but not pMOS-Chl1460, was observed in the presence of DNA obtained from clinical samples. It was supposedly linked with a higher sensitivity of Taq DNA-polymerase to the action of inhibitors in the synthesis of high-molecular DNA fragments. The observed high frequency of the inhibition (17%) of PCR makes it expedient to carry out this reaction with the use of the internal control.
Rar
V A
VA
Institute of Bioorganic Chemistry, Novosibirsk, Russia.
Maksimova
T G
TG
Morozova
O V
OV
Sartakova
M L
ML
Golovanova
O V
OV
D001483:Base Sequence; D002584:Cervix Uteri / Q000382:microbiology; D002692:Chlamydia trachomatis / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012150:Polymorphism, Restriction Fragment Length; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012015:Reference Standards
D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S
Ispol'zovanie vnutrennikh kontroleĭ razlichnoĭ dliny pri detektsii DNK Chlamydia trachomatis metodom polimeraznoĭ tsepnoĭ reaktsii.