Experience sharing of large-scale screening of new crown nucleic acid detection in high and high risk areas

From 20214 to 20222, Hunan Province supported Jilin nucleic acid testing team of 157 people to work and live in Changchun for a total of 40 days, completed 1223 million tubes of new crown nucleic acid testing, and served more than 10 million person-tim.

The main position of the author's work is the Guangzhou Jinyu "Falcon" dura membrane laboratory located in the Changchun Fifth Ring Gymnasi.

The nucleic acid test in Changchun is different from the pa.

One-zone reagent preparation

Reagent preparation is the first step in nucleic acid detection, and it is also the starting line for a complete and complete process, which determines the effectiveness of subsequent wo.

1 Time and process optimization suggestions

The daily testing task of the gas model laboratory is generally 40,000 to 80,000 tub.

Thawing the amplification reagents in advan.

The reaction solution and enzyme of the amplification reagent can be poured into a 50ml centrifuge tube at the same time according to the correct ratio for mixing, instead of mixing and mixing separate.

Use a fully automatic liquid workstation for automatic dispensing instead of using a discharge g.

2 Notes

The amount of reagent preparation should be as flush as possible with the detection amou.

The action is gentle and strictly prevent human-induced polluti.

Two-zone sample preparation

Sample preparation is the heaviest and most cumbersome area in nucleic acid testing, with many people, many posts, complicated equipment and crowded spa.

1 Time and process optimization suggestions

Due to the complexity of the positions, it is recommended that the personnel in each position are relatively fixed, which can reduce the error ra.

After the sample is received, it needs to be shaken and mixed, and the shaking time should be appropriately extended for 10-mixing 1 tube and 20-mixing 1 tu.

The beginning and end of each row of specimens should be marked and numbered with a marker p.

If the sample addition is wrong, if you can clearly know the wrong position of the sample addition, mark it clearly on the layout sheet; if it is not clear, the whole board needs to be redo.

After the sample addition is completed, the placement of the specimens: it can be placed separately according to the different sample.

Discarding of specimens: After uploading the results of the whole plate of specimens, the third district will issue an instruction after checking, and the personnel in the second district can discard the specimens (each plate of specimens is packaged and discarded separately, and the plate number is marked on the outside of the small yellow garbage ba.

If there is a reexamination specimen in a certain plate specimen, the plate specimen shall be temporarily stored until the reexamination is complet.

The re-examination needs to be laid out separately, and the positive re-examination specimens should not be added at the same time as possible in adjacent positio.

2 Notes

All reagents (lysing solution, washing solution, magnetic beads, eluent, e.

) in the nucleic acid extraction process should belong to the same batch number, and the plate number and sequence number should be written, and the marked positions should be in the same directi.

If there is no amplification result due to the wrong order of reagents, the entire plate needs to be redo.

Before using the extraction reagent, it is necessary to sample to check whether there is crystal formation at the bott.

The guanidine salt crystal can reduce the adsorption and release efficiency of the magnetic beads, resulting in the failure of nucleic acid extracti.

If the crystallization is caused by the temperature is too low, it needs to be rewarmed at 37 degrees Celsi.

For the crystallization caused by other reasons, the reagent needs to be replac.

After the extraction is completed, if it is found that there are too many residual magnetic beads in the magnetic rod sleeve and residual magnetic beads in the eluent, the extractor should be stopped and the instrument engineer should be contact.

Spotting and filming/covering posts, especially pay attention to the carry-over pollution caused by gloves, which need to be replaced or disinfected frequent.

If the sampler finds that there is no swab in the sampling tube, it will be processed as a refund (except environmental samplin.

The 96-well plate needs to be centrifuged for a short time before being loaded on the machi.

If it is stacked, it should be considered whether the centrifugal force will squeeze the film on the remote plate, resulting in a decrease in seali.

Three-region amplification and analysis

Since there may be many brands and models of PCR amplification instruments in the gas membrane laboratory, the staff in the amplification area should have a certain understanding of different PCR amplification instruments and be familiar with the operation before entering the cab.

Operators in the amplification area should be proficient in the principle of fluorescent PCR, the meaning of CT value, the adjustment of baseline, the meaning of negative and positive quality control, the interpretation of results, e.

, and be careful and cautio.

1 Time and process optimization suggestions

Positive or weak positive results need to be confirmed by two people and signed by two peop.

The layout sheet and the original sheet for each batch of review should be collected together and simply bound for easy verificati.

In the analysis part of the results at the bottom of the original sheet, the results of the board should be summarized, such as "full negative", "1 internal standard not checked", "1 double positive review", "1 single positive review", e.

, and sign for the results to be enter.

2 Notes

Pay attention to the fluorescence signal collection method of the amplification instrument, which is side scan or top sc.

For side scanning, opaque amplification tubes cannot be us.

Silicone covers or opaque/frosted octave covers cannot be used if the top is scann.

Before all amplification tubes are put on the machine, the tightness of the caps of each tube should be reconfirm.

After the amplification instrument is installed, the negative and positive quality control positions should be marked, and the remaining sample wells should be numbered, which is consistent with the layout she.

Try to open the thermal cover of the thermal cycler after the program ends and cool down to prevent the cover from bursti.

When analyzing the results, different reagents should pay special attention to which target gene each channel represents, and should not be confus.

When analyzing the results, if the amplification curve has a sloping line, a non-upright S-shaped curve, e.

, it is necessary to check the original curve, adjust the baseline, and re-analy.

When different reagents are used for the re-examination of the same tube of positive samples, the difference between the CT values ​​of the two needs to be within a reasonable range (calculated in advance according to the program setting method, sample volume, total volume, e.

If there is a very late single-gene take-off, no matter whether the instruction manual has been judged to be negative, a double-reagent review should be carried o.

In areas with many cases, the respiratory tract detoxification of patients varies greatly among individuals, and the single gene at the very back may be a small amount of detoxificati.

After the pollution is eliminated, it can be reported as suspicio.

Effectively control the temperature in the cab.

The heat dissipation of multiple amplification instruments is huge, and the damage to the amplification instruments due to excessive ambient temperature should be reduc.

Regional exchanges

Communication between districts is very importa.

The following is a summary of the matters that all districts need to pay attention to or invol.

Amplification reagents should be protected from ultraviolet radiation before and after spotting, and try to avoid light as much as possib.

The circulation of each plate of specimens should be carried at any time with a circulation she.

The circulation sheet should be marked with the plate number, the person who added the sample - the sample plate such as "XX-1", the extractor, the number of the extractor, the person on the amplification machine, The serial number of the amplification instrument, the result analyst and other informati.

Minimize unnecessary movement of people and goods between are.

Especially avoid the reverse flow of people and goo.

If flow is required, disinfection should be done to prevent product contaminati.

If there are multiple internal labels on a certain plate that cannot be found, first determine whether it is an environmental sample, and check the basic conditions of the sampling tu.

Then notify the second district to shake the sample again, replace the sampler and re-load the whole plate, replace the extractor for extraction, and replace the amplifier for amplificati.

If the situation still does not improve, you can notify the resampli.

If there are double-positive specimens in the initial screening, and the original tube becomes negative after re-examination, the original plate should be found and the whole plate should be re-examin.

75% alcohol can kill the new coronavirus and prevent biological hazards, but it cannot effectively prevent product contamination, and it is flammable and explosi.

Therefore, to prevent nucleic acid product contamination, use chlorine-containing disinfectant or nucleic acid scavenger as much as possib.

If a large number of specimens have ORF1ab or N gene tailing, the nucleic acid product contamination should be considered first, and the laboratory should suspend u.

Use chlorine-containing disinfectant or nucleic acid scavenger for complete sterilization, especially in some hidden locations, such as reagent storage boxes, refrigerator door handles, extractors and fully automatic dispensing workstation screens, guns,e.

Carry out multiple tests such as environmental sample testing in the laboratory and no-specimen blank control, and can be put into use again after there is no polluti.

The principles and methods of new crown nucleic acid detection are not complicated, but large-scale screening in high and high risk areas , strict prevention of contamination, and ensuring the accuracy of the results, there are many things that need to be paid attention .

The author only cites some of th.

Colleagues continue to discu.

There are still many deficiencies and imperfections, please experts and colleagues point o.

Expert Comments

Professor Nie Xinmin

Director of Laboratory Department, Third Xiangya Hospital, Central South University

The author of this article has been working on the front line of nucleic acid testing in large public hospita.

This year, he led a team to support nucleic acid testing in Anyang, Henan and Changchun, Jil.

He has a deep understanding of each step of nucleic acid testing, and is good at thinking and summarizi.

From the perspective of front-line nucleic acid testing personnel and quality managers , this paper analyzes the precautions that need to be paid attention to in large-scale nucleic acid testing in high and high risk areas, and proposes many solutio.

Many details pointed out in the article are often overlooked in work but are very critical, and it is worthy of reference and discussion by colleagues in the wo.

Professor Zhou Xiguo

Hunan Provincial Support Jilin Nucleic Acid Testing Team Chief Team Leader, Professor of Hunan Provincial Clinical Testing Center

In this epidemic in Changchun, Jilin Province, there were many positive samples, and quality control was difficu.

As one of the main quality leaders of the gas membrane laboratory, the author of this paper starts with process optimization and precautions, and points out the details that need to be paid attention to in all aspects of the nucleic acid detection operation, which can help colleagues in the future when encountering such problems in a timely manner Analyze the reasons and avoid detou.

This kind of experience summary and analysis is worthy of promoti.

I hope that colleagues can provide valuable opinions and gather the strengths of hundreds of schools to better defeat the new crown epidem.