[Quantitative competitive RT-PCR and quantitative detection of Escheriia coli acrA-mRNA].
A Quantitative detection assay of acrA-mRNA of Escherichia coli ATCC25922 was developed by Quantitative Competitive RT-PCR. Target Standard(TS) which was same as target-templete acrA was amplified by PCR with P1 and P2 as primers. Internal Standard (IS) which was shorter 68 bp then target-templete acrA was amplified by temperature-gradient-PCR with P1 and P3P2 as primers, whose annealing temperatures ranged from 55-65 degrees C, and the most suitable annealing temperature was acquired at 56 degrees C. Both TS and IS were largely amplified by PCR as above and extracted to store. Co-amplification with both TS and IS as templetes was optimized by temperature-gradient-PCR with P1 and P2 as primers, whose annealing temperatures were ranged from 55-65 degrees C, and then the most suitable annealing temperature was also acquired at 56 degrees C. Then co-amplification optimized as above was did again but with both cDNA of Escherichia coli (with target-templete acrA-cDNA copies unknown) and IS (10-fold serial dilution, and with IS copies known) as templates. The electrophoresis bands were photographed and analysed with UVI band and each band area was acquired, then linear regression analysis was did with SPSS ll.5 and CurveExpert l.3 and a competitive curve was drawn as y = -0.345 + 0.097x. Results revealed that the two kinds of product electrophoresis bands of co-amplification, whose templates were both 10-fold diluted IS and cDNA, could be distinguished clearly in 1.5% agarose gel because of 68bp discrepancy, and showed lighteness dimming gradually with IS copies 10-fold diluting. With the competitive curve, the copies of acrA-mRNA in sample could be counted accurately and easily.
Chen
Ai-mei
AM
Guangdong Institute of Microbiology, Guangzhou 510070, China. amchen1982@163.com
Chen
Yi-ben
YB
D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D008074:Lipoproteins / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000235:genetics*; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature
C083497:AcrA protein, E coli; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger