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Novel coronavirus nucleic acid detection

July 27, 2021

The nucleic acid detection has the characteristics of early diagnosis, high sensitivity and specificity, and is the "gold standard" for diagnosing new coronary pneumonia. Currently, the most widely used real-time fluorescent quantitative RT-PCR technology. Generally, the two targets located on the ORF1ab and N genes of the virus are detected. The same specimen must meet the double target positive or the repeated test as the single target positive, or the two specimens must meet the single target at the same time to confirm the positive of the SARS-CoV-2 virus nucleic acid.

 

1. Principle of nucleic acid detection kit? The unique gene sequence of the virus is used as the detection target. Through PCR amplification, the target DNA sequence we choose increases exponentially. Each amplified DNA sequence can be combined with a fluorescent-labeled probe that we added in advance. , Produce fluorescent signal, the more target genes amplified, the stronger the accumulated fluorescent signal. In samples without virus, since there is no target gene amplification, no increase in fluorescence signal can be detected. Therefore, nucleic acid detection is actually to determine whether there is viral nucleic acid in the sample by detecting the accumulation of fluorescent signals.

 

2. What are the types of nucleic acid testing samples?

Generally, nasal swabs, throat swabs, nasopharyngeal swabs, sputum, bronchial lavage fluid, alveolar lavage fluid and so on.

 

3. Five-step detection The new coronavirus detection procedure requires five steps: sampling, sample retention, storage, nucleic acid extraction, and computer testing. This requires rigorous scientific experiments to complete.

1. Wipe the back wall of the pharyngeal swab and bilateral pharyngeal tonsils 5-10 times each with a pharyngeal swab, and keep rotating the swab;

2. Medical personnel are required to keep the sample, and the swab head is immersed in the cell preservation solution, and the tail is broken. Tighten the tube cap immediately;

3. Save, put the sample tube in a sealed bag and send it for inspection in time, and the inspection process requires a strict transportation environment, stored at 2-8 degrees Celsius.

4. Operate nucleic acid extraction, extract nucleic acid from the inactivated virus sample for subsequent detection, and use automated equipment, such as a nucleic acid extractor.

5. Fluorescence PCR nucleic acid detection, that is, machine detection, it takes 70-80 minutes for the extract to undergo fluorescent PCR amplification reaction.

 

4. What are the problems with nucleic acid detection?

False negative, due to improper sampling, improper specimen storage, the use of different types of specimens, and the use of reagents from different manufacturers may cause the nucleic acid test results to be "false negative" and missed diagnosis;

High requirements for detection equipment or platforms, high sensitivity RT-PCR instruments are expensive, and require high laboratory cleanliness and operators;

Nucleic acid testing takes a long time, and it usually takes 4-6 hours to complete a RT-PCR test. However, considering sample transportation, In the case of a large backlog of samples, the results can usually be reported as soon as 24 hours. Therefore, when the nucleic acid test is negative, the addition of IgM and IgG antibody detection can make up for the shortcomings that the nucleic acid test is likely to cause missed diagnosis.