2. There are several positive results in a certain batch of test results, and the retest is still positive, but it is negative in other institutions. This result is mostly a strong positive in the sample, because the operator's improper operation leads to the subsequent samples are all positive, because it is the original sample pollution, so the retest is also positive. Specific process of nucleic acid detection in PCR Laboratory

The following is the specific process of nucleic acid detection in PCR laboratory, which can be used as a reference for qualified medical institutions to carry out virus nucleic acid detection.

1、 Personal level 3 protective clothing

Wear disposable hat, medical N95, disposable protective clothing, disposable shoe cover, disposable waterproof boot cover, one-layer latex gloves, outer disposable isolation clothing, protective eyepiece, second layer latex gloves, and enter the nucleic acid extraction area (Industrial nucleic acid extraction laboratory) as soon as possible after wearing.

2、 Sample inactivation treatment

Nucleic acid detection requires two staff members to quickly enter the laboratory for inactivation experiment. The biosafety cabinet should be equipped with absorbent cotton and disinfectant alcohol, and absorbent tablecloth if possible to deal with the overflow of samples in an emergency; open the secondary container, disinfect the surface with 75% alcohol, test the tightness of the sample tube, and then carry out the inactivation treatment. Under the inactivation conditions (56 ℃, 30min, constant temperature sample sterilizer and other methods), take out the alcohol inactivation tube, wipe the surface with alcohol, and remove it Put the samples into the biosafety cabinet.

3、 Manual nucleic acid extraction

Operators enter the reagent preparation area or separate clean laboratory.

1） Preparation of reagent area

According to the instructions, 130ml absolute ethanol was added to aw1, 160ml absolute ethanol was added to AW2. After adding absolute ethanol, the label was made in time. 310 μ l ave was added to 310 μ g carrier RNA and mixed upside down to make carrier RNA dissolve fully. Thus, the washing solution aw1, washing solution AW2 and carrier RNA were completed RNA configuration, and then transmitted to the sample processing area through the transfer window, or sent by human to the nucleic acid extraction laboratory.

2） Fragmentation of samples

A 1.5ml sterile EP tube was used to remove 560 μ l AVL lysate by pipette, 5.6 μ l carrier RNA was added, and 140 μ l inactivated sample of nucleic acid to be extracted was added. Vortex oscillation was performed for 15s, and the sample was still at room temperature for 10min. Put the EP tube into the centrifuge for instantaneous centrifugation to remove the pyrolysis products that may remain on the EP tube cover and prevent pollution. After pyrolysis, 560 μ l anhydrous ethanol was added, and the mixture was shaken for 15s. The EP tube was put into the centrifuge for instantaneous centrifugation to remove the pyrolysis products that may remain on the EP tube cover and prevent pollution.

630 μ l pyrolysis product was added to the centrifuge column and centrifuged at 8000 rpm for 1 min. if the centrifuge is outside the biosafety cabinet, the external surface of the centrifuge should be disinfected every time. To replace a new waste liquid collection tube, carefully remove the centrifuge column from the centrifuge, transfer the upper centrifuge column to the new collection tube, and continue to add 630 μ l of the remaining pyrolysis products into the centrifuge column. If the sample volume is greater than 140 μ L, repeat this step until all the pyrolysis products pass through the column and leave the center. Centrifuge at 8000 rpm for 1 min, and carefully transfer the centrifuge column to the new collection tube.

Add 500 μ L aw1 into the centrifuge column (note that the pipette suction head must contact the inner wall of the centrifuge column during the sample adding operation), and centrifuge at 8000 rpm for 1 min. Take out the centrifuge column carefully and replace it with a new one.

Take 500 μ L AW2 into the centrifugal column, centrifuge for 3 min at 14000 rpm, carefully take out the centrifugal column, replace with a new collecting tube at the upper end of the centrifugal column, place it in the centrifuge and centrifuge at high speed for 1 min, so as to ensure that the residual liquid in AW2 is completely centrifuged clean.

Take sterile 1.5ml EP tube to collect nucleic acid, put the upper end of the centrifuge column into 1.5ml EP tube carefully, take 40-60 μ l of eluent ave into the centrifuge column (Note: eluent should cover the membrane of the centrifuge column as much as possible), cover the centrifuge column with EP tube cover, and cut off the original centrifuge column cover with sterile scissors.

After standing at room temperature for 1 min, the nucleic acid was collected by centrifugation at 8000 rpm for 1 min. Discard the centrifugation column, extract nucleic acid in EP tube, mark and register.

4） Extraction of nucleic acid by instrument

First, prepare the nucleic acid extraction reagent according to the instructions, add 200 μ l inactivated sample to the nucleic acid extraction reagent, and according to the nucleic acid extraction instrument（ https://detail.1688.com/offer/641021810336.html?spm=a2615.7691456.autotrace-offerGeneral.13.7fce4754Hg7KEU ）After the operation of the instrument, the extraction of nucleic acid is completed and the nucleic acid is recovered.

5） PCR system configuration

According to the number of test samples, the prepared PCR system was fully mixed and centrifuged, and then sent to the sample processing nucleic acid sampling area or a separate sample sampling laboratory through the transfer window. According to 20 μ l reaction system packed in each well, 5 μ l nucleic acid or positive and negative control substance of sample to be tested were added in each well (please note that each detector contains 1 positive control and 3 negative controls, and the negative control should be randomly distributed), PCR tube should be sealed, and template sampling should be carried out in a separate biosafety cabinet if possible.

The staff will send the PCR tube to the PCR amplification area through the transmission window, or send it to the PCR amplification Laboratory for computer detection.

6） Computer test and result analysis

The staff enter the gene amplification analysis laboratory, take out the PCR reaction tube from the transfer window, mix and centrifuge the reaction system before operation, set the amplification parameters and analyze the interpretation results according to the instructions of the kit. When analyzing the results, strictly read the instructions, fully understand the sensitivity of the kit, methodological limitations and other comprehensive interpretation of the experimental results.

In order to prevent the contamination of PCR amplification, it is forbidden to open the cap of PCR amplification tube after the reaction and put it into the garbage can in the sealing belt.

7） Disinfection after detection

After the sample treatment, the staff should disinfect the outer gloves with 75% alcohol, take off the outer gloves and put them into the biological garbage can in the biosafety cabinet. After the test, 0.5-1% effective chlorine disinfectant or 75% ethanol should be used to wipe the surface and floor of biosafety cabinet. The medical waste generated in the safety cabinet should be sealed with three-layer garbage bags and put into the garbage can. The medical waste information should be marked on the garbage bags. At the end of the experiment, the biosafety cabinet and the laboratory should be disinfected by ultraviolet light for at least 30 minutes.

The experimental operator should have the 0.5-1% disinfectant or 75% ethanol spray disinfectant on the disposable isolating clothes, then remove the isolation clothing and put the medical waste into the garbage bin. After leaving the two level laboratory, the staff should remove the personal three class protective products in the buffer zone or the corridor. First, we should take the new outer gloves and remove the goggles. Put it in 75% ethanol for disinfection, turn it from top to bottom and from inside to outside, take off disposable protective clothing, outer gloves, disposable hat and shoe cover. Take off gloves, virus protective clothing need to be autoclaved. Before and after high pressure, medical waste should be strictly separated.

Virus related waste should be treated at 120 ℃ for 30 min with high pressure and humidity, and then treated as general medical waste.

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