Nucleic acid test method

The specific process of nucleic acid detection is as follows:

1. nucleic acid extraction

Use commercial reagents or equipment, such as silica gel column centrifugation, magnetic silica gel particle separation and automatic instruments, and follow the instructions. Attention should be paid to prevent RNA degradation when extracting RNA. DNA should be stored at -20℃, RNA and long-term DNA should be stored at -80℃.

2. cDNA synthesis by reverse transcription

Reverse transcription cDNA synthesis reaction needs to use reverse transcription primers, dNTPs, reverse transcriptase, RNase inhibitor, DTT, buffer, proper amount of ultrapure water without RNA/DNase and RNA template. The reverse transcription reaction is carried out in an amplification instrument or a water bath box at a specific temperature and time. It is suggested to use commercial RT-PCR one-step reagent for the first amplification reaction. Reverse transcription cDNA synthesis reaction needs to use reverse transcription primers, dNTPs, reverse transcriptase, RNase inhibitor, DTT, buffer, proper amount of ultrapure water without RNA/DNase and RNA template. The reverse transcription reaction is carried out in an amplification instrument or a water bath box at a specific temperature and time. The first round of amplification reaction was carried out using commercial RT-PCR one-step reagent.

3.PCR amplification reaction (nested PCR amplification method using secondary amplification)

PCR reaction needs primers, dNTPs, DNA polymerase (such as Taq enzyme, etc. ), buffer solution, proper amount of ultrapure water without RNA/dnase, and template (DNA or cDNA). In the amplifier, amplification is performed according to the set program. Nested PCR amplification method with secondary amplification was adopted.

4. Qualitative analysis of amplified products

The commonly used analysis method of amplified products is agarose gel electrophoresis, which is compared with the molecular weight standard to judge whether the amplified fragments are in the expected molecular weight range. Other amplification products analysis methods include restriction enzyme digestion analysis, specific probe hybridization analysis and DNA sequence analysis. Automatic nucleic acid amplification instrument uses enzyme-linked colorimetric analysis or fluorescent probe hybridization to determine.

5. Determine the results and complete the report

(1) experimental conditions: two positive controls and two negative controls should be made at the same time in each experiment. Only when the positive control amplified the expected fragment, the negative control did not amplify the fragment, and the results of the two parallel samples were consistent, the experiment could be established and the judgment of the positive or negative reaction results of nucleic acid could be made.

(2)HIV nucleic acid test positive: if nucleic acid positive reaction is found, samples should be collected repeatedly for re-examination; If the recheck result is positive, it is determined that the nucleic acid is positive; If the re-inspection result is negative, it is determined to be an uncertain result, and further follow-up testing is needed.

(3)HIV nucleic acid test is negative: only this experimental result can be negative.

(4) The test report shall be issued within 5 working days after the test is completed.