Inactivation at 56 u2103 may lead to degradation of viral nucleic acid
2019 the genetic material novel coronavirus is single stranded RNA. Therefore, the goal of researchers is to find the RNA of the new coronavirus in patients. This is also the gold standard of clinical diagnosis.
At present, fluorescent quantitative RT-PCR kit is mainly used in clinical detection. The method is to reverse the specific RNA sequence in the sample and amplify it. After more than 30 times of amplification, the virus gene fragment can reach a certain number for visual detection.
In theory, even if the template has only one virus, it could be detected. Zhao Qingshun said that in scientific research practice, when the number of templates (viruses) is greater than 100, the amplification results will be very stable.
However, when Zhao Qingshun saw a teaching video on the Internet, he couldnt help but wonder. The video was jointly produced by Beijing Union Medical College Hospital and Beijing Municipal Health Commission. Video 3:08-3:40 shows: before preparing the nucleic acid template, the collected samples should be inactivated for 30 minutes at 56 u2103.
A novel coronavirus pneumonia clinical laboratory test guide (trial version 2019) released by the Chinese Medical Associations laboratory medicine branch has also seen that before the amplification of nucleic acids, the specimen can be sterilized first. Including incubation at 56 u2103 for 30 minutes, adding protease K.
In the expert consensus novel coronavirus pneumonia nucleic acid detection issued by the inspection medicine branch of the Chinese Medical Association, it was clearly pointed out that the sample should be incubated at least 56 minutes or at least 45 minutes or higher to inactivate the sample.
The reporter confirmed through the interview that the vast majority of laboratory doctors were in accordance with the above specifications, and carried out virus inactivation at 56 u2103 for different times, and then prepared the nucleic acid template.
The inactivation treatment is carried out for the consideration of biological safety
For the sake of biological safety, the inactivation treatment is carried out to protect the staff engaged in the detection from being infected by the virus. Zhao Qingshun said.
The novel coronavirus novel coronavirus laboratory guidelines for the diagnosis of new type coronavirus (Third Edition) were published by Peking Union Medical College Hospital, and the guidelines for prevention and expert consensus issued by the Chinese Medical Associations laboratory medicine branch were based on relevant documents of the National Health Service Commission, including the new coronavirus laboratory biosafety guidelines (Second Edition).
However, the reporter inquired the relevant documents of the national health and Health Commission, which did not make clear the specific methods of virus inactivation, but generally required: infectious materials or live virus nucleic acid detection after inactivation by reliable methods.
Zhao Qingshun further consulted the nucleic acid detection instructions issued by the CDC, the school of public health of the University of Hong Kong and BGI, and did not find the step of 56 u2103 inactivation.
How much does the degradation of viral nucleic acid affect the detection results
According to the opinion of the disease control department, the laboratory medicine branch of the Chinese Medical Association and the laboratory doctors of the relevant hospitals, it will not have a great impact on the test results. According to Zhao Qingshun and other experts, high temperature treatment of human samples before nucleic acid extraction may be one of the important reasons for the high false negative rate of nucleic acid detection of new coronavirus.
Zhao Qingshun studied the effect of high temperature inactivation on the detectable amount of porcine epidemic diarrhea virus (a kind of coronavirus from live vaccine). The results showed that the samples stored in the ordinary isotonic solution (Hanks solution) were incubated at 56 u2103 for 30 minutes, resulting in a half reduction of the detectable coronavirus template in the samples. If the samples were incubated at 92 u2103 for 5 minutes, the detectable amount of coronavirus would be reduced The loss of coronavirus template was more than 96%.
Theoretically, it is not ruled out that the target RNA fragment of the new coronavirus is not easy to be degraded in the high temperature inactivation above 56 u2103 Zhao Qingshun said frankly, but I prefer my judgment to be wrong, and I dont want a false negative test result due to some details that are not considered properly.
In addition, different brands of sample preservation solution will also have a greater impact on the test results. In the experiment, Zhao Qingshun found that the detectable amount of viral nucleic acid was three times of that of the control group (Hanks solution) when the samples of porcine epidemic diarrhea virus were stored in R503 storage solution developed by Nanjing nuoweizan at 56 u2103 for 30 minutes, and 42 times of that of the control group if they were inactivated at 92 u2103 for 5 minutes.
In response to the high false negative rate of nucleic acid detection, Professor Wang Chengbin, chairman of the branch of laboratory medicine of the Chinese Medical Association, also pointed out that different extraction reagents may have differences in the quantity and quality of the final extracted nucleic acid, thus directly affecting the detection results. He suggested that for some highly suspected cases or cases whose test results are difficult to determine, more than two reagents should be used for testing and verification.
False negative means missed test, which will not only lead to the clinical diagnosis of suspected patients can not be rapid, but also make the missed test become a potential source of virus infection. Zhao Qingshun put forward suggestions for this. First, try to use a sample collection tube without ribonuclease pollution. Second, place the sample in a sample preservation solution that can protect the viral nucleic acid from high temperature inactivation, so as to ensure that the sample RNA can be protected in the whole process from preservation, transportation to high temperature inactivation, so as to ensure the quality of nucleic acid for clinical detection as much as possible, and reduce the detection of viral nucleic acid The template was artificially damaged before nucleic acid extraction.