Abstract: Objective: To evaluate the ability of triple Taqman probe real-time PCR to the detection and differentiation of vanA, vanB and vanM genotypes of Vancomycin-resistant enterococci (VRE). METHODS: Choosing the culture method and PCR-sequencing assay as the reference standard，respectively, a total of 254 VRE and 90 strains of no-VRE were conducted by triple Taqman probe real-time PCR collected from 2005 to 2015 in China.
Results: When detect the VRE strains, the sensitivity, specificity, PPV and NPV of triple Taqman probe real-time PCR were 99.6%, 100%, 100% and 98.9%, respectively. When differentiate among three genotypes of vanA, vanB and vanM VRE, the sensitivity, specificity, PPV and NPV of the triple Taqman probe real-time PCR were …show more content…
Single colony ,obtained from agar culture, were inoculated onto medium with teicoplanin 32μg / mL and medium with vancomycin 6μg / mL medium, respectively. Read the test results after 24 hours. The strains which can grow on both medium are defined as VanA phenotype VRE.While the strains which can not grow on teicoplanin-containing medium, but grows on vancomycin-containing medium are defined as VanB phenotype …show more content…
After boiling, centrifuge the bacteria liquid 30sec with 12000r and take the supernatant as template for PCR-sequencing and real-time PCR.
The procedure was carried out according to the triple Taqman probe real-time PCR kit detection instructions. 25.5ul reaction system consist of 20ul reaction solution, 0.5ul reaction enzyme, 5ul template. The reaction conditions, sequence of primer and probe of Taqman real-time PCR method are listed in Table 1. In each batch, the known genotype of vanA, vanB, vanM strains are used as positive control, ATCC29212 and saline are used as negative control and blank control, respectively.
20 μL vanA, vanB, vanM primer reaction system consists of 1 μL upstream primer, 1 μL downstream primer, 10 μL 2 ofTaq PCR MasterMix (KT201), 6 μL sterile ultrapure water and 2 μL DNA template. VanA, vanB, vanM primer N-VAN and other reaction sequences and reaction procedures are listed in Table 2.All PCR products were sent to sequencing. Each batch of reactions contained a negative control, a positive control, a blank control, which were identical to the triple Taqman probe real-time PCR
Results: When detect the VRE strains, the sensitivity, specificity, PPV and NPV of triple Taqman probe real-time PCR were 99.6%, 100%, 100% and 98.9%, respectively. When differentiate among three genotypes of vanA, vanB and vanM VRE, the sensitivity, specificity, PPV and NPV of the triple Taqman probe real-time PCR were …show more content…
Single colony ,obtained from agar culture, were inoculated onto medium with teicoplanin 32μg / mL and medium with vancomycin 6μg / mL medium, respectively. Read the test results after 24 hours. The strains which can grow on both medium are defined as VanA phenotype VRE.While the strains which can not grow on teicoplanin-containing medium, but grows on vancomycin-containing medium are defined as VanB phenotype …show more content…
After boiling, centrifuge the bacteria liquid 30sec with 12000r and take the supernatant as template for PCR-sequencing and real-time PCR.
The procedure was carried out according to the triple Taqman probe real-time PCR kit detection instructions. 25.5ul reaction system consist of 20ul reaction solution, 0.5ul reaction enzyme, 5ul template. The reaction conditions, sequence of primer and probe of Taqman real-time PCR method are listed in Table 1. In each batch, the known genotype of vanA, vanB, vanM strains are used as positive control, ATCC29212 and saline are used as negative control and blank control, respectively.
20 μL vanA, vanB, vanM primer reaction system consists of 1 μL upstream primer, 1 μL downstream primer, 10 μL 2 ofTaq PCR MasterMix (KT201), 6 μL sterile ultrapure water and 2 μL DNA template. VanA, vanB, vanM primer N-VAN and other reaction sequences and reaction procedures are listed in Table 2.All PCR products were sent to sequencing. Each batch of reactions contained a negative control, a positive control, a blank control, which were identical to the triple Taqman probe real-time PCR