New Coronavirus nucleic acid detection steps

New Coronavirus nucleic acid detection steps

1、 The specific operations are as follows

1. Virus sampling:

Take the patient’s saliva sample or nasopharyngeal flocked swab sample, and save the virus sampling tube.Virus preservation solutions commonly used in disposable virus tubes include inactivated and non inactivated types. The following are two models of virus transport medium:

1) Inactivated type: the inactivated virus transport medium characterized by killing the virus and retaining nucleic acid fragments. Application:detect COVID-19, influenza virus, hfmv and influenza A.

2) Non inactivated type: the non inactivated virus transport medium does not contain lysate. It can retain the activity and integrity of pathogens.Application:virus culture and reproduction to make the detection results more accurate.

flocked swab specimen collection_New Coronavirus nucleic acid detection steps

2. Nucleic acid extraction:

Extract the genetic material from the patient’s sleeping fluid or nasopharyngeal swab sample. If a patient carries a virus, there will be RNA of the virus’s genetic material in the sample. Sampling personnel should pay attention to prevent RNA degradation when extracting RNA. At the same time, the extracted DNA shall be stored at – 20 ℃, and the RNA and DNA requiring long-term storage shall be stored at – 70 ℃ or liquid nitrogen.

3. Reverse transcription synthesis of cDNA:

reverse transcription of RNA in the extract, reverse transcription of RNA into cDNA, reverse transcription of cDNA synthesis reaction, using reverse transcription primers, dNTPs, reverse transcriptase, RNase inhibitor and DTT. Buffer, an appropriate amount of ultrapure water without RNA / DNase and RNA template. We propose to use commercially available RT-PCR one-step reagent for one round amplification of di.

4. PCR amplification reaction (nested PCR amplification method with secondary amplification):

Use specific primers of virus cDNA for PCR amplification in an amplification instrument. Nested PCR amplification using secondary amplification.

5. Result analysis and judgment:

If the DNA band of the virus is amplified, it is determined that the virus exists in the patient; If no DNA band is amplified, it is determined that the sample taken by the disease has no virus.

2、 Experimental precautions:

① We should set up two positive controls and two negative controls for each test. Only when the positive control enlarges the expected fragment, the negative control does not enlarge any fragment, and the results of two parallel samples are consistent.We can determine the nucleic acid positive or negative reaction results.

② Nucleic acid test is positive: if nucleic acid is positive, we should repeatedly collect samples for retest. If the retest result is nucleic acid positive, we can confirm that the sample result is nucleic acid positive. If the retest result is nucleic acid negative, the nucleic acid test result is uncertain.