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- W2374553244 abstract "Objective To establish a method for quantitative detection of cytotoxin associated gene A (cagA) positive Helicobacter pylori (cagA + Hp) by competitive polymerase chain reaction. Methods The lactose regulatory protein (LacI) specific combining sequence (the lactose operator, 21 bp) was inserted into the cagA fragment (fcagA, 400 bp) by overlapping extension PCR. By cloning lactose regulatory gene (lacI) into pGEX 4T 3, transforming E.coli JM109 with pGEX 4T 3/lacI, and inducing the bacterium with isopropyl β D thiogalactopyranoside (IPTG), we got the active fusion protein of glutathione s transferase (GST) and LacI. In the competitive PCR, rfcagA was used as the internal standard template and pMC3 that contained most part of cagA from the 5′ end or Hp cagA as the competitive one. Since one primer of the PCR was labelled with biotin at the 5′ end, the PCR products could conjugate to the microplate wall that coated with avidin. When the GST LacI was added to the walls, only the products containing the lactose operator sequence could combine with it, and the GST could catalyze 1 chloro 2,4 dinitrobenzene and glutathione to form a substance that had the maximal ultraviolet(340nm) absorbance. Results When the copies of the internal standard was fixed (100 copies), a standard curve of A 340 against the logarithmic value of the competitive templates was established. Using this method, we found that, among the 14 bacteria cultural media, 9 were cagA positive with the copies from 6.3×10 10 /L to 2.14× 10 11 /L. The coincidentce rate was 100%. Conclusion This is a specific,sensitive and applicable method for quantitative detection of cagA." @default.
- W2374553244 created "2016-06-24" @default.
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- W2374553244 date "2000-01-01" @default.
- W2374553244 modified "2023-09-23" @default.
- W2374553244 title "Quantitative detection of cagA of helicobacter pylori by competitive PCR" @default.
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