Is the abnormal PCR amplification curve caused by the virus transport media?
In the nucleic acid detection of the new coronavirus, the very key technology is the real-time fluorescent quantitative PCR experiment of nucleic acid, which is the core technology of the new coronavirus detection. So what effect does the virus transport media used for virus sampling joints have on the PCR amplification of nucleic acids? This article makes a brief discussion.
Does the virus transport media affect nucleic acid PCR amplification?
Judging the result from the PCR amplification curve:
The fluorescent quantitative PCR instrument in the new crown detection has become a routine equipment for molecular biology research. The rapid development of this detection method and the urgency of the detection task have also put forward higher requirements for the detection personnel, requiring them to be proficient in judging the results of the data. For the judgment of the result of fluorescence quantitative PCR, the most intuitive judgment is to see whether the amplification curve is normal. In normal experiments, you will encounter problems with abnormal amplification curves, such as discounted amplification curves, poor repeatability between multiple wells, and uplifted amplification curves of negative samples.
Reasons for abnormal PCR amplification curve:
1. Confirm whether the software settings are correct. Check the kit instructions to check whether the time, temperature, cycle number, fluorescence collection, etc. are set correctly, and whether the selected reagent contains ROX as a reference fluorescent dye. Some results show that the amplification curve of the multi-component graph is not uplifted, which is obviously a negative sample, but the amplification graph curve is uplifted, so that it crosses the threshold line, and instead has a CT value. This is because the software makes a mistake in the automatic deduction of the baseline. The method of manually adjusting the baseline can be normal by redefining the baseline.
2. Make sure that the consumables and instrument accessories are used correctly. Consumables are more important for real-time PCR. Many abnormal amplification curves are caused by improper use of consumables.
3. To determine whether the reagents used are normal, first determine whether the reagents are effective, including checking whether the reagents are within the validity period, whether they are repeatedly frozen and thawed many times, and whether the transportation conditions are normal. If all of the above are normal, then you have to consider whether there is any negligence in the operation details.
From the above points, it can be seen that the virus transport media will not directly affect the amplification curve, it will only affect the virus samples, but this can be done through a control experiment with positive samples to determine whether the virus preservation solution has an impact on the virus samples. There are two types of Desheng virus transport media, both the inactivated type and the activated type are suitable for nucleic acid PCR experiments.
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