Academic journal article Iranian Journal of Public Health

Evaluation of the Analytical Sensitivity of Loop-Mediated Isothermal Amplification (Lamp) Method for Rapid Detection of Extended Spectrum Beta Lactamases, Ctx-M-1 Family

Academic journal article Iranian Journal of Public Health

Evaluation of the Analytical Sensitivity of Loop-Mediated Isothermal Amplification (Lamp) Method for Rapid Detection of Extended Spectrum Beta Lactamases, Ctx-M-1 Family

Article excerpt

Background: In this study LAMP, a new molecular tech- nique was evaluated for detection of CTX-M 1 gene.

Methods: Sequences of related gene of CTX-M-1 family were retrieved from Gene Bank and aligned. By using of the consensus sequence, LAMP specific primers by Eiken primer design online software were designed. Using the outer pri- mers F3 and B3, the target gene in a strain producing CTX- M-1 was amplified and the product was cloned in E coli Top10 F' host. Confirming the recombinant plasmid ob- tained (pTZ-CTX-M1), its concentration determined by spec- trophotometry. Tenfold serial dilution of the plasmid was prepared and subjected to evaluate by CTX-M1-LAMP method at standard conditions in presence of the designed primers (F3, B3, FIP, BIP). Amplification of the target se- quence was detected by using Loopamp Real Time Turbi- dimeter LA-320C device and direct observation of generated turbidity.

Results: When using the turbidimeter, the last dilution of the plasmid that showed the proliferation, was 0.5395 fg or 161 copies of the CTX-M-1 gene. So the value was determined as the limit of detection of CTX-M1-LAMP method. The detec- tion limit of the method when using direct observation of turbidity was calculated 5.395 fg or 1610 copies. …

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