The CycleavePCR™ O157(VT1/VT2) Detection Kit is designed to detect Enterohemorrhagic E. coli (EHEC) which causes hemorrhagic colitis with accompanying melena and severe abdominal pain, and in addition, hemolytic uremic syndrome. This kit is for detection via real-time PCR amplification of verotoxin genes (VT1 and VT2).
This kit employs Cycling Probe Technology (CPT) for detection, which is a highly sensitive detection method utilizing a combination of a chimeric probe (composed of RNA and DNA), and RNase H. Although the chimeric probe is similar to TaqMan®-style probes, it differs by containing a small fragment of RNA which gets cut by the RNase H when the probe forms a hybrid with the complementary sequence. This cleavage results in the emission of strong fluorescence. If there is a mismatch within the probe-binding region, the probe will not be cut by RNase H. In this way, the CPT allows highly specific detection which can recognize even a single nucleotide polymorphism.
This is a multiplex kit where probes are labeled with two different dyes: one with FAM for detecting VT1 and the other one with Cy3 which detects VT2. During amplification, our sample will then emit fluorescence depending upon the genes present. For example, if VT1 is present, we will get fluorescence in the FAM channel; if VT2 is present, we will get signal in the Cy3 channel. If the sample is positive for both genes, we get emission in both the channels. In addition, we get a signal for IC (Internal Control, provided with the kit) which acts as a amplification control; IC signal emits in the Cy5 channel.
The kit components are Takara Ex Taq R-PCR 5 units/µL (0.25 µL per reaction), Tli RnaseH II (0.5 µL per reaction), 5X reaction mixture which includes dNTPs and the internal control (5 µL per reaction), VT primer mix (2 µL per reaction), VT chimeric probe mix (5 µL per reaction), VT1 and VT2 positive controls whose concentration is 1x104 copies/µL (1 µL per reaction).
The kit is very easy to use and the the technical insert very nicely explains how to set the temperature on the SmartCycler®, how to prepare the mastermix and how to analyze the results, complete with a troubleshooting chart. The preparation of the reagent mix is as follows: 5 µL 5X reaction mix, 2 µL Primer mix, 5 µL VT chimera probe mix, 0.25 µL Takara Ex taq R-PCR, 0.5 µL Tli RNase H II, 2 µL Template (1 µL of PC) and 10.25 µL PCR grade water to make the final volume 25 µL.
The general protocol which we use with this kit as follows: Initial hold at 95şC for 10 seconds. Stage 2 comprises 3 temperature cycles: initial denaturation at 95şC for 5 seconds, annealing at 55şC for 10 seconds (optics ON) and finally, extension at 72şC for 15 seconds. We generally obtain the results in 1 hr, 20 min. Our main application is detection of DNA extracted from various samples, including environmental, clinical and dairy food product samples.
| Comment on this Review |
Comments? Questions? Discuss this review of CycleavePCR™ O157(VT1/VT2) Detection Kit Ver.2.0 From Takara with the author of this review, Tarun Jain, and the rest of the Biocompare community.
|
Tarun Jain
Product Specialist
Biotron
Molecular Diagnostics
India
more articles...
