What Is the Procedure for a HIV PCR Test?

HIV nucleic acid test, which can directly detect HIV RNA, can detect HIV infection before detecting serological changes, and is more sensitive than the P24 antigen detection method.

HIV nucleic acid test

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Qualitative HIV nucleic acid test

1.1 Methods and reagents

1.1.1 Method

(1) The detection methods are commercial kits and laboratory self-built methods. Commercial reagents should be operated strictly in accordance with the instructions. The method described below is a self-built method for reference.

(2) The reverse transcription PCR (RT-PCR) method is used to detect plasma or serum samples. It is recommended to use the RT-PCR one-step method for the first round of PCR amplification; the PCR method is used to detect blood cells or tissue samples. A nested PCR method with two rounds of amplification is generally used.

(3) Set up positive, negative and blank controls. The positive control is a sample that is homogeneous with the test sample and contains the target gene; the negative control is a sample that is homogeneous with the test sample and does not contain the target gene. The positive and negative control samples should be in accordance with the processing procedures of the sample to be tested. The number of negative controls should be set in different positions according to the number of experimental samples.

1.1.2 Reagents

(1) PCR primers: Primers such as amplified HIV gag and / or pol and / or env and / or LTR are generally used. For reverse transcription of RNA, downstream specific primers or random primers can be used. Primer design can refer to the literature or design by yourself. Common HIV epidemic strains should be covered as far as possible, and degenerate primers can also be used.

(2) Main reagents: including reagents required for nucleic acid extraction and purification, reverse transcription, and PCR. Use commercial nucleic acid extraction and purification reagents, reverse transcriptase reaction reagents and PCR amplification reagents.

(3) Anti-pollution reagents: The self-built test method in the laboratory can use anti-pollution reagents, refer to the uracil DNA glycosylase anti-pollution method.

1.2 Amplify the target gene fragment

1.2.1 Collection and processing of samples

1.2.2 Nucleic acid extraction

Commercially available reagents or equipment such as silica gel column centrifugation, magnetic silica particle separation methods, and automated instruments can be used and operated in accordance with the instructions. Care should be taken to prevent RNA degradation when extracting RNA. DNA should be stored at -20 ° C. RNA and long-term storage DNA should be stored at -80 ° C.

1.2.3 Reverse Transcription Synthesis of cDNA

Use commercial reagents and follow the instructions. Reverse transcription cDNA synthesis requires the use of reverse transcription primers, dNTPs, reverse transcriptase, RNase inhibitors, DTT, buffers, and appropriate amounts of RNA / DNase-free ultrapure water and RNA templates. Perform reverse transcription reaction in the amplifier or water bath at the specified temperature and time. It is recommended to use commercial RT-PCR one-step reagents for the first round of amplification reactions.

1.2.4 PCR amplification reaction

Use commercial reagents according to the instructions. PCR reactions require primers, dNTPs, DNA polymerases (such as Taq enzymes, etc.), buffers, appropriate amounts of RNA / DNase-free ultrapure water, and templates (DNA or cDNA). In the instrument, amplification was performed according to a set program. Generally, a nested PCR amplification method using secondary amplification is used.

1.3 Amplification Product Analysis and Results Report

1.3.1 Analysis of amplified products

A common method for analyzing amplified products is agarose gel electrophoresis, which is compared with molecular weight standards to determine whether the amplified fragment is within the expected molecular weight range. Other amplification product analysis methods include restriction enzyme digestion analysis, specific probe hybridization analysis, and DNA sequence analysis. The automated nucleic acid amplification instrument uses the principle of enzyme-linked colorimetric analysis or fluorescent probe hybridization.

1.3.2 Results determination and reporting

(1) Conditions for the establishment of the experiment: two positive controls and two negative controls must be made for each test. Only the positive control amplified the expected fragment, the negative control did not amplify any fragments, and the results of the duplicate samples were consistent. Under the circumstances, the experiment is established, and the result of nucleic acid positive or negative reaction can be determined.

(2) Positive HIV nucleic acid test: using commercial testing reagents, if a nucleic acid positive reaction is found, samples should be repeatedly collected for retesting. If the retest result is a nucleic acid positive reaction, it is judged as a nucleic acid positive, and the retest result is a nucleic acid negative reaction, it is judged as The results are uncertain, and further follow-up testing is required.

(3) Negative HIV nucleic acid test: Only report negative results in this experiment.

(4) The test report should be issued within 7 working days after the test is completed.

HIV nucleic acid quantitative test

2.1 Method

HIV nucleic acid quantitative detection is mainly based on two methods: target nucleic acid amplification RT-PCR and signal amplification. Among the commonly used methods in China, NucliSens Esay Q HIV-1 v1.1 uses international units (IU / ml), and its relationship with the copy number of NASBA is basically 1: 1; the copy number of Amplicor Cobas is about 1.2 to 1.5 international units; bDNA The copy number of the method is approximately 0.8 to 1.0 IU. The relationship between different methods is related to HIV subtypes.

2.2 Reagents

Commercial reagents registered and approved by China Pharmaceutical and Biological Products Administration must be used and operated strictly in accordance with the instructions.

2.2.1 Target nucleic acid amplification reagents and performance

(1) RT-PCR amplification reagent

1) Amplicor HIV-1 Monitor v1.5 (RT-PCR microplate capture colorimetric analysis method), manual extraction of nucleic acids (isopropanol precipitation), colorimetric analysis. The target nucleic acid amplification site is the gag gene region, which can amplify the AK gene subtype of the HIV-1M group. The standard detection method has a linear range of 400 to 750,000; the ultrasensitive method has a linear range of 50 to 100,000 RNA copies / ml.

2) COBAS Amplicor HIV-1 Monitor v1.5, manual extraction of nucleic acids (isopropanol precipitation), instrument determination. The target nucleic acid amplification site is the gag gene region, which can amplify the AH gene subtype of the HIV-1M group. The standard detection method has a linear range of 400 to 750,000; the ultrasensitive method has a linear range of 50 to 100,000 RNA copies / ml.

3) COBAS AmpliPrep / COBAS Amplicor HIV-1 Monitor v1.5 (COBAS Amplicor Analyzer), the instrument extracts nucleic acids, and the instrument determines. The target nucleic acid amplification site is the gag gene region, which can amplify the AH gene subtype of the HIV-1M group. The standard method has a linear detection range of 400 to 1,000,000; the ultrasensitive method has a linear range of 50 to 100,000 RNA copies / ml.

The above three reagents are based on the PCR amplification target nucleic acid detection method, which differs only in the use of equipment, the degree of automation, and the method of sample preparation.

4) COBAS AmpliPrep / COBAS TaqMan HIV-1 Test (real-time fluorescent probe RT-PCR analysis method), instrument extraction of nucleic acids, real-time fluorescent probe PCR amplification instrument determination. The target nucleic acid amplification site is the gag gene region, which can amplify the AH gene subtype of the HIV-1M group. The detection linear range is 40 to 10,000 RNA copies / ml, which is wider than the detection linear range of the above three methods. The sample can be detected without dilution once.

The above four reagents use one internal quantitative standard, one negative, one weak positive and one strong positive external external control. All use dUTP / UNG anti-contamination reagents.

5) LCx HIV RNA Quantitative Assay (competitive RT-PCR microparticle enzyme immunoassay), manual or instrument extraction of nucleic acids (activated silica gel column purification), instrument determination. The target nucleic acid amplification site is the pol (integrin) gene region, which can amplify the AG subtype of HIV-1 gene M group and group 0 virus, and especially detect the M group C gene subtype and some recombinant viruses. The detection linear range is 50 to 1,000,000 RNA copies / ml; one internal quantitation standard and six external quantification standards are used.

6) RealTime HIV-1 Assay (RT-PCR analysis with real-time fluorescent probe), using a unique double-stranded fluorescent probe. Nucleic acid extraction (purification of magnetic particles) by hand or instrument, instrument determination. The position of the amplified target nucleic acid is the pol (integrin) gene region, and the amplified HIV-1 gene subtype is the AG subtype of group M and group 0 and the virus of group N. The detection linear range is 40 to 1,000,000 RNA copies / ml. Use an internal quantitation standard. The test results are highly correlated with LCx HIV RNA Quantitative Assay results.

(2) Based on nucleic acid sequence amplification reagents (NASBA amplification technology): HIV-1 RNA is directly amplified in an isothermal manner.

NucliSens Esay Q HIV-1 v1.1 (Real-Time Fluorescent Probe Assay), uses fluorescently labeled molecular beacon probe technology to detect amplicons. Nucleic acid extraction by hand or instrument (silica gel purification, isopropanol precipitation), instrument determination. The amplified target nucleic acid position is the gag gene region, and the amplified HIV-1 gene subtype is the AG subtype of group M. The detection linear range is 50 to 3,000,000 RNA international units / ml; an internal quantitative standard is used, and there are no negative external external quality controls. Each time an integer multiple of 8 is tested, up to 48 samples. The test results are highly correlated with the results of the Versant HIV-1 RNA and Amplicor HIV-1 Monitor v1.5 methods. V2.0 kits have been recommended internationally.

2.2.2 Signal Amplification Reagents and Performance

Versant HIV-1 RNA 3.0.bDNA (branched DNA probe hybrid microtiter plate capture colorimetric analysis), using the principle of multistage signal amplification of branched DNA. No RNA purification and PCR amplification steps are required. Virion was concentrated by centrifugation and the virus RNA was released by digestion with detergent and proteinase K to measure the virus. The position of the amplified target nucleic acid is the pol gene region, and the amplified HIV-1 gene subtype is the AH subtype of group M. The detection linear range is 50 to 500,000 RNA copies / ml; six external quantitative standards are used, one Negative, 1 weak positive, and 1 strong positive external external control. An integer multiple of 12 samples can be detected at a time.

All the above reagents can use EDTA and ACD as the anticoagulant of the sample. NucliSens HIV-1 QT and NucliSens Esay Q HIV-1 v1.1 and Versant HIV-1 RNA 3.0.bDNA reagent can also use heparin as an anticoagulant. If heparin-treated plasma samples must be used for RT-PCR amplification, heparin must be digested directly with heparinase.

2.2.3 Real-time PCR technology

Real-time fluorescent quantitative PCR technology refers to the method of adding a fluorophore to a PCR reaction system, using fluorescence signal accumulation to monitor the entire PCR process in real time, and finally performing a quantitative analysis of an unknown template through a standard curve. There is a linear relationship between the number of cycles Ct when the fluorescence signal in each reaction tube reaches a set domain value and the logarithm of the starting copy number of the template. A standard curve can be made using a standard with a known starting copy number, where the abscissa represents the logarithm of the starting copy number and the ordinate represents the Ct value). Therefore, as long as the Ct value of the unknown sample is obtained, the initial copy number of the sample can be calculated from the standard curve.

The experimental detection process of real-time quantitative PCR amplification can be divided into: (1) sample preparation, extraction and concentration of target RNA molecules, and removal of possible inhibitors. (2) Real-time PCR. Fluorescently labeled oligonucleotide probes are used to detect PCR products. The detection principle is based on the fluorescence signal growth curve and the number of cycles. (3) RT-PCR reaction. Viral RNA uses reverse transcriptase to reverse transcribe RNA into cDNA, and then amplifies specific fragments by DNA polymerase. (4) Detection of amplified products. Based on the setting of the detection threshold, when the viral load is high, the fluorescence signal can be detected at a low cycle number, and when the viral load is low, fluorescence can be detected at a high cycle number. The number of cycles is linear with the sample load. Quantitative detection of sample load can be made by using the standard to make a standard curve of the number of cycles and load.

Pool nucleic acid qualitative detection (Pooling PCR)

Using collective nucleic acid amplification detection techniques and methods, and utilizing the high sensitivity of nucleic acid detection methods, collective nucleic acid detection can be performed on samples that are highly suspected to be infected and antibody-negative to detect window-infected patients in time. This method is more cost-effective than nucleic acid detection in a single sample.

3.1 Sample assembly procedure

3.1.1 According to the amount of pre-processed samples, calculate the number of pre-formed primary and secondary collections, and record the primary and secondary collections and the corresponding original sample numbers on the registration form.

3.1.2 Pipette a 130mL sample and transfer it into a centrifuge tube labeled with the secondary collection; 10 samples form a 1300mL secondary collection sample, and vortex and mix thoroughly.

3.1.3 Aspirate 210mL samples from the five secondary collection tubes, transfer them into the centrifuge tube labeled with the primary collection, and form a primary collection sample consisting of 50 samples in a volume of 1050mL, and vortex and mix thoroughly.

3.1.4 Aspirate 500mL collection samples from each of the primary and secondary collection tubes, aliquot them into another correspondingly labeled centrifuge tube, and use ultrasensitive nucleic acid detection reagents for detection.

3.1.5 Preparation of negative collection external quality control products: Use 50 HIV antibody and nucleic acid negative samples, and collect 5 negative secondary collection external quality control products and 1 primary collection external quality control product respectively according to the above steps.

3.1.6 Preparation of external quality control for positive collection: From 9 HIV antibody and nucleic acid negative samples and one positive sample containing at least HIV RNA10c / ml, transfer 130mL to the centrifuge tube to form a 1300mL positive secondary collection External quality control. From the 4 prepared negative secondary collection external quality control products and the positive secondary collection external quality control product, 210 mL was transferred to the centrifuge tube labeled as the primary positive collection external quality control product to form a Grade positive collection of external quality controls.

3.1.7 The primary and secondary negative and positive collection external quality controls are used for the detection of primary and secondary collection samples in each round of RT-PCR.

3.2 Detection and decomposition route of aggregate samples

Use of commercial nucleic acid detection reagents should be performed in strict accordance with the reagent instructions. The detection is performed according to the PCR protocol of each commercial nucleic acid reagent set, and the number of collected samples is operated according to each reagent protocol. The method is briefly described as follows:

3.2.1 The HIV RNA RT-PCR hypersensitivity detection method is used to detect the primary collection sample, and the positive primary collection sample enters the next detection step.

3.2.2 The HIV RNA RT-PCR hypersensitivity detection method is used to detect all the secondary collection samples that make up the positive primary collection, and the positive secondary collection samples enter the next detection step.

3.2.3 The HIV RNA RT-PCR standard detection method is used to detect all 10 individual samples that make up a positive secondary collection sample to determine a single sample that is positive for nucleic acid.

Infant HIV infection nucleic acid test

Infants and babies born to HIV-infected mothers can use HIV nucleic acid (DNA or RNA) tests to diagnose early HIV infection within 18 months of birth. Although HIV RNA detection sensitivity is higher in the early stages of infection (within 1 month after birth), HIV DNA testing is not affected by maternal perinatal antiretroviral therapy, antiretroviral drugs in human milk, and preventive resistance in infants and young children Interference with retroviral therapy affects early diagnosis. In addition, considering mother's blood contamination, cord blood is not recommended for HIV nucleic acid testing. For the procedure and results of antibody testing in infants and young children, see Chapter 2 (HIV antibody testing).

4.1 Application

Infants and young children born to HIV-infected women under 18 months of age; infants and young children under 18 months of age have unknown HIV status of their mothers, HIV-related clinical manifestations in children, and clinically suspected HIV infection

4.2 Test procedure and result report

4.2.1 Collect the first blood sample (blood sample can be prepared as DBS or EDTA anticoagulated whole blood) 6 weeks (42 days) after the baby is born, and submit it for inspection.

4.2.2 If the first blood sample is tested positive, collect a second blood sample for testing as soon as possible. If both blood samples show a positive response, report "positive results of early diagnosis of HIV infection in infants" to diagnose HIV infection in children. Follow-up and condition monitoring of HIV-infected children in a timely manner, refer them to children's antiviral treatment medical services, and provide services such as opportunistic infection prevention; if the second blood sample is negative, wait until the baby is full Blood samples were taken again for testing at 3 months. If the first blood sample is negative, continue to provide child care and follow-up services, and wait for the baby to collect blood samples for testing after 3 months.

4.2.3 If the infant has a negative test again after 3 months, report Infant HIV diagnostic test results are negative, and treat as uninfected children, and continue to provide child health follow-up services. The HIV antibody detection process for children born to HIV-infected mothers "(Figure 4) begins HIV antibody testing and finally determines the status of child infection. If the baby has a positive test again after 3 months, take a blood sample for testing as soon as possible. The third blood sample tested positive and reports positive early diagnosis of HIV infection in infants; if the third blood sample tested negative, reports infant early diagnosis of HIV infection negative; provided in accordance with the aforementioned procedures Corresponding services.

4.2.4 Breastfeeding children who have a negative response to the "Early Diagnosis of Infant HIV Infection" test at different times should stop breastfeeding for 6 weeks and 3 months (if the test result is positive as soon as possible, as soon as possible at 6 weeks) ) Collect blood again for qualitative nucleic acid detection and early diagnosis. After 18 months, children can be tested directly for antibodies.

4.2.5 If the baby is three months old but less than 12 months old when the first blood is collected, two blood samples should be collected at different times as soon as possible; at the same time, the two blood samples should be submitted for testing and tested according to the aforementioned process. If it is 12 months since the child's first blood collection, the HIV antibody test should first be performed in accordance with the "HIV antibody test procedure for children born to HIV-infected mothers" (Figure 4). If the HIV antibody test reagents produced by two different principles or by the manufacturer have negative test results, the child is excluded; if the HIV antibody test reagents have a positive test result, and the infection status of the child cannot be determined through antibody testing, they can be collected at different times Two blood samples were tested for "early diagnosis of HIV infection in infants" according to the aforementioned procedure. If a child is 18 months old when the first blood is drawn, the HIV antibody test should be performed according to the HIV antibody test procedure (Figure 1), without the need for an "early diagnosis of HIV infection in the baby" test.

HIV nucleic acid testing currently has nucleic acid DNA and nucleic acid RNA.

HIV nucleic acid testing should be performed in a suitable laboratory. Standard operating instructions and strict management systems related to experiments and laboratory management should be established. Staff must fully understand the various regulations and strictly implement them.

Laboratory partitions and functions

The laboratory should set up two separate working areas: the pre-nucleic acid amplification area and the post-nucleic acid amplification area. Preamplification

Including reagent preparation area and sample processing area, located in different rooms or areas; nucleic acid amplification area including amplification area and amplification product analysis area, located in different rooms or areas. The functions of each zone are:

1.1 Reagent preparation area: storage, preparation and dispensing of amplification reagents.

1.2 Sample processing area: sample registration, processing, and packaging; nucleic acid extraction, storage, and use; first round of PCR amplification.

1.3 Amplified region: nucleic acid amplification. If the second round of nested PCR is performed in this zone, it should be performed inside a PCR shield.

1.4 Amplification product analysis area: electrophoresis, hybridization, imaging, sequence determination, result analysis, registration and reporting of amplification products.

Provided that the following requirements are met, the pre- or post-nucleic acid amplification zone can be located in one room:

1.4.1 Pre-nucleic acid amplification area There are two experimental areas in the laboratory. The reagents are configured in a clean bench and the sample processing is performed in a biological safety cabinet.

1.4.2 When a fully enclosed amplification and detection system is used in the post-nucleic acid amplification area, such as a real-time fluorescent PCR instrument.

1.4.3 Each experimenter uses its own reagents, consumables, pipettes, and containers for contaminants.

1.4.4 Clean and disinfect the operating area and shared appliances before and after the experiment.

1.4.5 Reagents, appliances, instruments and equipment in each area are dedicated to that area and must not be used crosswise.

.Lab staff and requirements

Personnel conducting HIV nucleic acid testing must be qualified to work in AIDS testing laboratories, and they must have received provincial or higher laboratory operation techniques, AIDS laboratory biosafety training, and training from manufacturers.

.Laboratory facilities and equipment

3.1 Equip corresponding facilities and equipment according to the testing items.

3.2 The facilities and equipment in each area are dedicated and should be configured in the corresponding area according to the experimental requirements.

3.3 Reagent preparation area: equipped with refrigerator, ultra-clean bench, ordinary centrifuge, sampler, shaker, waste container, removable UV lamp.

3.4 Sample processing area: -80 ° C refrigerator, biological safety cabinet, high-speed refrigerated centrifuge, sampler, oscillator, ice maker or refrigeration module, constant temperature water bath or dry bath, water supply and drainage equipment, waste container, UV lamp .

3.5 Amplification area: Configure a nucleic acid amplification instrument, a general refrigerator, a laminar-free PCR operation cabinet or a clean bench, a microcentrifuge, a sampler, a waste container, and a UV lamp.

3.6 Amplification product analysis area: equipped with electrophoresis apparatus, electrophoresis tank, sampler, UV transmission instrument, photography / imaging equipment, ordinary refrigerator, ordinary centrifuge, constant temperature water bath, microwave oven, water supply and drainage equipment, waste container, ultraviolet lamp . The sequencer can be placed in other specialized experimental areas.

3.7 Special or disposable work clothes, hats, masks, shoe covers and cuffs should be used in all areas, equipped with UV-resistant glasses.

Laboratory biosafety

Must meet the general biosafety requirements of AIDS laboratories. 5. Measures to prevent nucleic acid residue pollution 5.1 Strictly implement the laboratory zoning system to prevent laboratory nucleic acid residue (Carry-over) pollution measures

5.2 Each area is only used for specific operations and must not be engaged in other tasks.

5.3 Special system for instruments and materials

Instruments, equipment, materials and facilities shall be marked according to the work area, and shall not be mixed.

5.4 One-way work flow system

5.4.1 The airflow in the laboratory shall flow from the pre-amplification zone to the post-amplification zone, and shall not flow in the reverse direction. It is recommended to maintain weak positive pressure in the pre-amplification region and weak negative pressure in the post-amplification region.

5.4.2 The one-way work flow direction of the experimenter should be: reagent preparation area sample processing area amplification area amplification product analysis area, no reverse flow is allowed. In principle, experimenters cannot return to the pre-amplification area for work on the same day after working in the post-amplification area.

5.4.3 The one-way working flow direction of experimental supplies should be: reagent preparation area sample processing area amplification area amplification product analysis area, and no reverse flow is allowed. Experimental supplies include experimental materials (reagents, samples, and amplification products), experimental equipment (containers, plates, lab clothes, hats, masks, gloves, shoe covers), office supplies (recording paper, pens, etc.), cleaning materials, etc. .

5.5 Laboratory treatment procedures to prevent nucleic acid residue contamination

5.5.1 Before the experiment, apply 70% ethanol or 0.1 1% sodium hypochlorite solution or the surface of the operation table or utensil, and wipe it with a paper towel after two minutes.

5.5.2 Before the experiment, move in the required reagents, consumables, samples, and containers for pollutants.

5.5.3 All reagents, consumables, pipettes, etc. used for qualitative and quantitative nucleic acid detection must be used separately. After the experiment, remove the personal items to the storage area; close the pollutant container and put it in the dirt bag; take out the common utensils to the storage area; apply 70% ethanol or 0.1 to 1% sodium hypochlorite solution on the surface of the operation table or utensil. Wipe with a paper towel after a few minutes; turn on the UV lamp for at least 1 hour.

5.5.4 Regularly (weekly) comprehensively clean the laboratory. Clean all work surfaces, floors, and equipment surfaces in the room with 70% ethanol or 0.1 to 1% sodium hypochlorite. Use ultraviolet lamps to disinfect the table and air. Clean the pipette regularly (monthly).

5.5.5 Periodically (monthly) monitor and report the status of residual nucleic acid contamination in the laboratory. The monitoring sites include the laboratories and shared corridors in the area before and after nucleic acid detection.

5.5.6 After each test, the DNA sequence of the same batch of test samples and the DNA sequence of the previous batch of test samples shall be analyzed for genetic evolution tree contamination.

Waste disposal system

6.1 All waste should be treated as HIV contaminated items.

6.2 Chemicals such as ethidium bromide should be processed according to the relevant protocol.

Early diagnosis

Early diagnosis of infants: Infants born to HIV-infected mothers less than 18 months of age can be diagnosed with two positive HIV nucleic acid tests at different times. Children over 18 months of age have the same diagnosis as adults: a diagnosis of acute HIV infection syndrome or epidemiological history and two positive HIV nucleic acid tests at different times.

Multi-sample collection methods can also be used to collect nucleic acid testing of raw blood plasma and blood samples from high-risk populations with negative HIV antibodies in blood collection and supply institutions to detect window infections in time, reduce "residual risk", and reduce second-generation transmission.

Assistant diagnosis of difficult samples

In general, HIV antibody testing is sufficient to make a correct diagnosis of HIV infection. However, in special cases, the results of a simple HIV antibody test cannot make a clear diagnosis, such as when the antibody is uncertain in the early stages of infection or the end of the disease. RNA measurements can help provide evidence of early or end-stage HIV infection. Due to the sensitivity of the reagents, after receiving effective antiviral treatment, and a small number of infected persons with long-term low-level virus, other test data and sample background conditions should be comprehensively judged.

Genetic variation monitoring

It can be used for HIV molecular epidemiological surveillance, including differential diagnosis of HIV-1 and HIV-2 infections, analysis of the transmission chain of HIV infection, identification and analysis of HIV genotypes and recombinant viruses, and monitoring of trends in HIV genetic variation in the population.

Drug resistance surveillance

It can be used for HIV resistance detection and monitoring, to guide public health doctors to understand the epidemic situation of drug-resistant virus strains, and to guide clinicians to judge the effectiveness of antiviral treatment and modify antiviral treatment plans.

Course monitoring and prediction

There is a certain pattern of changes in viral load after HIV infection, and this change is closely related to the course of the disease. Therefore, regular viral load testing can help determine the stage of disease development and determine appropriate treatment options.

The relationship between HIV viral load and 6-year incidence is: 5.4% at viral load <500c / ml; 16.6% at 501 to 3,000c / ml, and 3,000 to 10,000c / ml 31.7%; 55.2% at 10,000 to 30,000 c / ml; 80% at> 30,000 c / ml. When the CD4 + T cell count of HIV-infected persons is less than 200 / l, the viral load and the risk of developing AIDS within 3 to 6 months are: 4.9% when the viral load is less than 10,000 c / ml; 10,000 12.7% at 29,999c / ml; 17.7% at 30,000 99,990c / ml; 22.4% at> 100,000 c / ml.

Guide antiviral therapy and determine efficacy

Antiviral therapy usually shows a good therapeutic effect after the viral load reaches a certain level (such as> 35,000 to 50,000c / ml). After treatment, the detection of the virus level can determine whether the treatment is effective. It is usually considered clinically effective to reduce viral levels by more than 0.5 log after 6 months of treatment.

Quality control should consider the management process in each step of the entire process from sample reception to report issuance, including: (1) personnel; (2) laboratory partition and environment; (3) equipment; (4) testing process (reagents, operation process , For external quality control products).

All relevant inspectors need to be trained in operations, be able to operate independently and skillfully, pass the assessment, and hold a certificate to work.

1 Laboratory zoning and environment

The HIV-1 viral load testing laboratory should, in principle, be divided into three separate work areas: reagent preparation area, sample processing area, and amplification product analysis area, and should be located in different rooms. The air flow direction of the three zones is strictly required: from the reagent preparation zone to the sample processing zone, and then to the amplification product analysis zone, the flow cannot be reversed.

2 Instrument and equipment quality control

The sampler and temperature and humidity meter must be calibrated by the metrology department once a year; the HIV-1 viral load detector, real-time fluorescent PCR instrument, and centrifuge can be commissioned by the company to calibrate once a year. Measure the temperature of refrigerators and water baths with a calibrated thermometer and make a record.

3 Quality control of testing process

Ensure that all kits are valid and registered with the State Food and Drug Administration, use DNA and RNase-free water; strictly follow the Standard Operating Procedures (SOPs) of instruments and reagents, and do not modify them without authorization. Each experiment requires the use of an external quality control in the kit and an external external quality control with HIV-1 RNA of 5000 to 15000 copies / ml; each experiment uses the external quality provided by the kit according to the instructions of the kit Controls and meet external quality control requirements.

4 External quality control

The National AIDS Reference Laboratory organizes a proficiency testing program for viral load twice a year, with 5 samples each time.

Butcher A, Spadoro J: Using PCR for detection of HIV-1 Infection. Clin Imm Newsletter, 1992, 12: 73-76.

Guidelines for the Use of Antiretroviral HIV-Infected Adults and Adolescents MMWR Recommendations and Reports April 24, 1998/47 (RR-5); 42-82.

Bayer HIV-1 RNA3.0 Assay (bDNA) Operation manual Rev. 2006-07

Roche AMPLICOR HIV MONITOR TEST Procedure Manual, version 1.5

Nuclisens & reg; EasyQHIV-1 Operation Manual, V1.0-2, 2002-05

Fractions of HIV-1 Seropositive Persons by Two Nucleic Acid Amplification Assays, AIDS Research and Human Retrovirus 1993, 9: 259-265.

"Guidelines for HIV-1 Viral Load Determination and Quality Assurance (Trial)" ((China Centers for Disease Control and Prevention, February 2008)

National AIDS Testing Technical Specifications (2009 Revision)