All DNA extracts were screened in triplicate 10μL PCRs to detect the presence of DNA from pathogenic Leptospira spp. using the lipL32 TaqMan real-time PCR assay [1 (link)] containing the following: 1x TaqMan Environmental PCR master mix (Applied Biosystems, Foster City, CA, USA), 0.9μM of each primer, 0.45μM of the MGB probe, and 1μl undiluted DNA template. Quantitative PCRs were run interchangeably on Applied Biosystems QuantStudio (QS) QS7 and QS12 Real-Time PCR Systems with QS 12K Flex or QS Real-Time PCR software, as appropriate, under the following conditions: 50°C for 2 minutes, 95°C for 10 minutes, and 45 cycles of 95°C for 15 seconds and 58°C for 1 minute. Positive (L. interrogans strain Fiocruz L1-130) and non template controls were included on all runs. LipL32 amplicons from all positive samples were subjected to direct Sanger sequencing to confirm that the amplified product was Leptospira, using the same forward and reverse primers for the PCR (all replicates from a single sample were sequenced independently). Treatment and sequencing conditions are described below.
Taqman environmental pcr master mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TaqMan Environmental PCR master mix is a ready-to-use solution designed for real-time PCR applications in environmental testing. It contains all the necessary components, including DNA polymerase, nucleotides, and buffer, to perform the PCR reaction.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using taqman environmental pcr master mix
1
Detecting Pathogenic Leptospira via qPCR
2
Pathogenic Leptospira Detection by qPCR
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All DNA extracts were screened in triplicate 10µL PCRs to detect the presence of DNA from pathogenic Leptospira spp. using the lipL32 TaqMan® real-time PCR assay (4) containing the following: 1x TaqMan® Environmental PCR master mix (Applied Biosystems, Foster City, CA, USA), 0.9µM of each primer, 0.45µM of the MGB probe, and 1µl undiluted DNA template.
Quantitative PCRs were run interchangeably on Applied Biosystems QuantStudio® (QS) QS7 and QS12 Real-Time PCR Systems with QS 12K Flex or QS Real-Time PCR software, as appropriate, under the following conditions: 50°C for 2 minutes, 95°C for 10 minutes, and 45 cycles of 95°C for 15 seconds and 58°C for 1 minute. Positive (L. interrogans strain Fiocruz L1-130) and non template controls were included on all runs. LipL32 amplicons from all positive samples were subjected to direct Sanger sequencing to confirm that the amplified product was Leptospira, using the same forward and reverse primers for the PCR (all replicates from a single sample were sequenced independently). Treatment and sequencing conditions are described below.
Quantitative PCRs were run interchangeably on Applied Biosystems QuantStudio® (QS) QS7 and QS12 Real-Time PCR Systems with QS 12K Flex or QS Real-Time PCR software, as appropriate, under the following conditions: 50°C for 2 minutes, 95°C for 10 minutes, and 45 cycles of 95°C for 15 seconds and 58°C for 1 minute. Positive (L. interrogans strain Fiocruz L1-130) and non template controls were included on all runs. LipL32 amplicons from all positive samples were subjected to direct Sanger sequencing to confirm that the amplified product was Leptospira, using the same forward and reverse primers for the PCR (all replicates from a single sample were sequenced independently). Treatment and sequencing conditions are described below.
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