Why the false-negative rate of nucleic acid tests in new crown pneumonia is so high
Experts say virus nucleic acid is not detected due to degradation
Our reporter Zhang Ye
Since the outbreak of New Coronary Pneumonia, the topic of excessive false-negative nucleic acid detection has been the focus of attention of all parties. It has been reported that the positive rate of detection of new coronavirus using fluorescent quantitative RT-PCR as a detection method is currently only 30% -50%, resulting in a surprisingly high false negative rate.
There are many reasons for the false negative rate. On February 22, Zhao Qingshun, a professor of developmental biology and genetics at the Institute of Model Animals of Nanjing University, accepted an exclusive interview with reporters from the Science and Technology Daily. According to guidelines issued by relevant institutions, the collected samples should be inactivated at 56 ° C before nucleic acid detection. It is very likely that the nucleic acid of the new coronavirus will be degraded, which will cause it to not be detected normally, and eventually increase the false negative rate. The research result "High-temperature inactivation process before viral nucleic acid extraction significantly reduces the amount of detectable viral nucleic acid template" has been pre-released on the scientific paper platform of the Chinese Academy of Sciences.
56 ° C inactivation may cause degradation of viral nucleic acids
The genetic material of the 2019 new coronavirus is single-stranded RNA. Therefore, the goal of the researchers is to find the RNA of the new coronavirus in patients. This is also the gold standard for clinical diagnosis.
At present, the clinical detection mainly uses the fluorescent quantitative RT-PCR kit. The method is to amplify a specific RNA sequence in a specimen after reverse transcription, and after more than 30 amplifications, the viral gene fragments can be visually detected after reaching a certain number.
"In theory, even if there is only one virus in the template, it may be detected." Zhao Qingshun said, in the practice of scientific research, when the amount of template (virus) is greater than 100, the amplification result will be very stable.
However, when Zhao Qingshun saw a teaching video on the Internet, he couldn't help worrying. The video was jointly produced by Peking Union Medical College Hospital and Beijing Municipal Health Commission. The video 3 minutes 08 seconds to 3 minutes 40 seconds shows that before preparing the nucleic acid template, the collected samples need to be inactivated at 56 ° C for 30 minutes.
The reporter also saw in the "Guidelines for Biosafety Protection of New Coronavirus Pneumonia Clinical Laboratory Testing (First Trial Edition) 2019" issued by the Laboratory Medicine Branch of the Chinese Medical Association: before nucleic acid amplification, specimens can be disinfected first. Include incubation at 56 ° C for 30 minutes and add proteinase K.
In the Expert Consensus for Detection of New Coronavirus Pneumonia Virus Nucleic Acids issued by the Chinese Medical Association Laboratory Medicine Branch, it was clearly stated that samples need to be incubated at 56 ° C for at least 45 minutes or higher for inactivation.
The reporter confirmed through interviews that most of the laboratory physicians performed virus inactivation at 56 ° C for a period of time in accordance with the above specifications before preparing nucleic acid templates.
The problem with this is that viral RNA is easily degraded by ribonuclease, because this enzyme is most active at 60 ° C. The ribonuclease comes from two aspects, one is inside the sample cell, and the other is the foreign pollutants during the collection, preservation and transportation.
The inactivation process is carried out for biosafety considerations
"The inactivation process is for the sake of biosafety, and to protect the workers who carry out the test from being infected by the virus." Zhao Qingshun said.
It is for this reason that the relevant documents issued by the National Health Commission explicitly require inactivation of specimens.
The teaching videos produced by Peking Union Medical College Hospital and the "Guidelines for Protection" and "Expert Consensus" issued by the Chinese Medical Association Laboratory Medicine Branch are based on relevant documents from the National Health and Medical Commission, including the "Guidelines for Biosafety of New Coronavirus Laboratories (second Edition) "" Technical Guide for Laboratory Testing of New Coronavirus Pneumonia (Third Edition) "and so on.
However, the reporter consulted the relevant documents of the National Health and Health Commission, which did not make clear the specific method of virus inactivation, but only general requirements: nucleic acid detection of infectious materials or live viruses after reliable inactivation .
Zhao Qingshun further checked the nucleic acid testing instructions issued by the Centers for Disease Control and Prevention, the School of Public Health of the University of Hong Kong, and BGI, and did not find the 56 ° C inactivation step.
How much impact does viral nucleic acid degradation have on test results
The reporter found in interviews that different people had clear answers to this question:
The opinion from the disease control department, the laboratory medical branch of the Chinese Medical Association, and the test physicians of the relevant hospitals is that it will not have a great impact on the test results. And Zhao Qingshun and other experts believe that high-temperature anti-virus treatment of human samples before nucleic acid extraction may be one of the important reasons for the high false-negative rate of New Coronavirus nucleic acid detection.
Zhao Qingshun carried out a study on the effect of high temperature inactivation on the detectable amount of the virus using the swine epidemic diarrhea virus (a coronavirus from a live vaccine) as a model. The results showed that the samples stored in ordinary isotonic solution (Hank's solution) After 30 minutes incubation at 56 ° C, the detectable coronavirus template in the sample was reduced by half. If incubated at 92 ° C for 5 minutes, the detectable coronavirus template was lost by more than 96%.
"Theoretically, it is not ruled out that the target RNA fragment of the New Coronavirus cannot be easily degraded in the high temperature inactivation above 56 ° C." Zhao Qingshun said frankly, "but I would rather my judgment is wrong, and I don't want to consider it because of some details. Results in false negative results. "
In addition, different brands of sample storage solution will also have a greater impact on the test results. Zhao Qingshun found in experiments that when the swine epidemic diarrhea virus sample was stored in R503 preservation solution developed by Nanjing Novizan at 56 ° C for 30 minutes, the detectable amount of viral nucleic acid was the detection amount of the control group (Hank's solution). 3 times; and if it is inactivated at 92 ° C for 5 minutes, the detection amount is 42 times that of the control group.
Professor Wang Chengbin, chairman of the Chinese Medical Association Laboratory Medicine Branch, also pointed out that in response to the high false negative rate of nucleic acid detection, the number and quality of nucleic acids extracted by different extraction reagents may be different, which directly affects the test results. He suggested that for some highly suspect cases or cases where the test results are difficult to determine, it is recommended to use more than two reagents for testing and verification.
"False negatives mean missed tests, which will not only lead to the inability to quickly diagnose suspect patients in the clinic, but also make the missed testers a potential source of viral infection." Zhao Qingshun made recommendations for this. The sample collection tube, the second is to place the specimen in a sample preservation solution that can protect the viral nucleic acid from high temperature inactivation, so as to ensure that the sample RNA is fully protected from storage, transportation, and high temperature inactivation. Detection of nucleic acid quality, reducing artificial damage to viral nucleic acid detectable templates before nucleic acid extraction.