1.1 Methods and reagents
1.1.1 Methods
(1) The assay methods are commercialized kits and laboratory-constructed methods, and the commercialized reagents should be operated strictly according to the instructions. The method described below is the laboratory self-constructed method for reference.
(2) Detection of plasma or serum samples using reverse transcription PCR (RT-PCR) method, it is recommended that the first round of PCR amplification using RT-PCR one-step method; detection of blood cells or tissue samples using PCR method. Generally use the set PCR method with two rounds of amplification.
(3) Establishment of positive, negative and blank controls. Positive controls are samples that are homogeneous with the sample to be tested and contain the target gene; negative controls are samples that are homogeneous with the sample to be tested and do not contain the target gene. Positive and negative control samples should be consistent with the processing procedures of the samples 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: generally use primers for amplification of HIVgag and/or pol and/or env and/or LTR. Downstream specific primers or random primers can be used for RNA reverse transcription. Primer design can be based on the literature or self-designed, and should cover as many common HIV strains as possible, and concurrent primers can also be used.
(2) Main reagents: These include the 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: laboratory self-constructed detection methods can use anti-pollution reagents, refer to uracil DNA glycosylase anti-pollution methods.
1.2 Amplification of target gene fragments
1.2.1 Sample collection and processing
1.2.2 Nucleic acid extraction
Commercialized reagents or equipment such as silica gel column centrifugation, magnetic silica gel pellet separation methods, and automated instrumentation can be used and operated according to the instructions. When extracting RNA, care should be taken to prevent RNA degradation, and DNA should be stored at -20℃, while RNA and DNA for long-term storage should be stored at -80℃.
1.2.3 Reverse transcription cDNA synthesis
Use commercial reagents and follow the instructions. The reverse transcription cDNA synthesis reaction requires the use of reverse transcription primers, dNTPs, reverse transcriptase, RNAase inhibitor, DTT, buffer and appropriate amounts of RNA/DNAase-free ultrapure water and RNA template. Perform the reverse transcription reaction in an 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 reaction.
1.2.4 PCR Amplification Reactions
Use commercial reagents and follow the instructions. PCR reactions require primers, dNTPs, DNA polymerase (e.g., Taq enzyme, etc.), buffer, and an appropriate amount of RNA/DNA enzyme-free ultrapure water, as well as template (DNA or cDNA). In the amplifier, amplification is performed according to a set program. A secondary amplification suite PCR amplification method is typically used.
1.3 Amplification Product Analysis and Reporting of Results
1.3.1 Amplification Product Analysis
A common method for analyzing amplification products is agarose gel electrophoresis, which is used to compare with a molecular weight standard and to determine whether or not the amplified fragments are within the expected molecular weight range. Other methods of amplification product analysis include restriction endonuclease digestion, specific probe hybridization, and DNA sequence analysis. Automated nucleic acid amplifiers use enzyme-linked colorimetric analysis or fluorescent probe hybridization and other principles of determination.
1.3.2 Result determination and reporting
(1) The conditions for the establishment of the experiment: each test needs to be done at the same time as the two positive controls, two negative controls, only if the positive control amplifies the expected fragments, the negative control does not amplify any fragments, and the results of the two parallel samples are the same, then the experiment is established, and the results of the nucleic acids can be made to determine the results of positive or negative reactions.
(2) HIV positive nucleic acid test: the use of commercial testing reagents, found that the nucleic acid positive reaction, should be repeated collection of samples for retesting, retesting the results of the nucleic acid positive reaction is determined as nucleic acid positive, retesting the results of the nucleic acid negative reaction is judged to be an inconclusive result, the need for further follow-up testing.
(3) Negative HIV nucleic acid test: only the result of this experiment can be reported as negative.
(4) The test report should be issued within 7 working days after completion of the test. 2.1 Methods
The quantitative detection of HIV nucleic acid is mainly based on two methods: target nucleic acid amplification RT-PCR and signal amplification amplification. Among the commonly used methods in China, NucliSens Esay Q HIV-1 v1.1 uses international units (IU/ml), and the copy number relationship with NASBA is basically 1:1; the copy number of Amplicor Cobas is about 1.2 to 1.5 international units; and the copy number of bDNA method is about 0.8 to 1.0 international units. The relationship between the different methods is related to the subtype of HIV.
2.2 Reagents
Commercialized reagents that have been registered and approved by the Chinese Drug and Biological Products Supervision Administration must be used and operated in strict accordance with the instructions.
2.2.1 Target nucleic acid amplification reagents and performance
(1) RT-PCR amplification reagents
1) Amplicor HIV-1 Monitor v1.5 (RT-PCR microtiter plate capture colorimetric assay), nucleic acid extraction by hand (isopropanol precipitation), colorimetric assay determination. The target nucleic acid location for amplification is the gag gene region, which amplifies the A-H genotypes of the HIV-1M group. The linear range of the assay for the standard method is 400-750,000; the linear range for the ultrasensitive method is 50-100,000 RNA copies/mL.
2) COBAS Amplicor HIV-1 Monitor v1.5, Nucleic acids were extracted manually (isopropanol precipitation) and measured instrumentally. The target nucleic acid location for amplification is the gag gene region, which amplifies the A-H genotype subtypes of the HIV-1M group. The linear range of the assay for the standard method is 400-750,000; the linear range for the ultrasensitive method is 50-100,000 RNA copies/mL.
3) COBAS AmpliPrep/COBAS Amplicor HIV-1 Monitor v1.5 (COBAS Amplicor Analyzer), instrumental extraction of nucleic acids, instrumental measurement. The target nucleic acid is the gag gene region, which amplifies the A-H genotypes of the HIV-1M group. The linear range of the standard method is 400-1,000,000; the linear range of the ultrasensitive method is 50-100,000 RNA copies/mL.
All three of these reagents use PCR amplification target nucleic acid-based assays, differing 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 assay), the nucleic acid is extracted by the instrument, and measured by the real-time fluorescent probe PCR amplification instrument. The target nucleic acid position is the gag gene region, which can amplify the A-H genotypes of HIV-1M group. The linear range of the assay is 40-10,000,000 RNA copies/mL, which is wider than that of the above three methods, and the assay can be completed in one run without dilution of the sample.
All four reagents use one internal quantitative standard, one negative, one weak positive and one strong positive external external quality control. All use dUTP/UNG anti-contamination reagents.
5) LCx HIV RNA Quantitative Assay (competitive RT-PCR microparticle enzyme immunoassay),Nucleic acids were extracted manually or instrumentally (activated silica gel column purification), and instrumentally determined. The target nucleic acid is the pol (integrase) gene region, which can amplify HIV-1 group M subtypes A-G and group 0 viruses, especially group M subtype C and some recombinant viruses. The linear range of the assay is 50 to 1,000,000 RNA copies/mL; one internal and six external quantitation standards are used.
6) RealTime HIV-1 Assay (real-time fluorescent probe RT-PCR assay), using a unique double-stranded fluorescent probe. Nucleic acids are extracted manually or instrumentally (magnetic particle purification) and instrumentally determined. The target nucleic acid location is the pol (integrase) gene region, and the HIV-1 genotypes amplified are group M subtypes A-G, group 0 and group N viruses. The linear range of the assay is 40 to 1,000,000 RNA copies/mL. An internal quantitative standard was used. Assay results were highly correlated with LCx HIV RNA Quantitative Assay results.
(2) Nucleic Acid Sequence-Based Amplification Reagent (NASBA Amplification Technology): Direct amplification of HIV-1 RNA in an isothermal manner.
NucliSens Esay Q HIV-1 v1.1 (Real-Time Fluorescent Probe Assay), detects the amplicon using fluorescently labeled molecular beacon probe technology. Nucleic acids are extracted manually or instrumentally (silica gel purification, isopropanol precipitation) and measured instrumentally. The target nucleic acid is the gag gene region, and the HIV-1 subtype is the A-G subtype of group M. The linear range of the assay is 50-3. The linear range of the assay was 50 to 3,000,000 RNA international units per milliliter; an internal quantitative standard was used, and there were no negative or negative external quality controls. Integer multiples of 8 samples were tested up to 48 at a time. Results are highly correlated with those of Versant HIV-1 RNA and Amplicor HIV-1 Monitor v1.5. The V2.0 kit has been recommended internationally.
2.2.2 Signal Amplification Reagents and Performance
Versant HIV-1 RNA 3.0.bDNA (Branched DNA Probe Hybridization Microtiter Plate Capture Colorimetric Assay), utilizes the principle of multistage signal amplification by branched DNA. No RNA purification and PCR amplification steps are required. Centrifugation concentrates the virion and digestion of the virus with detergent and proteinase K releases the viral RNA, which is measured instrumentally. The target nucleic acid location for amplification is the pol gene region, and the amplified HIV-1 genotype is the A-H subtype of group M. The linear range of the assay is 50 to 500,000 RNA copies/mL; six external quantitation standards are used, with one negative, one weakly positive, and one strongly positive external external quality control. An integer multiple of 12 samples can be tested at a time.
All of the above reagents can use EDTA and ACD as anticoagulants for the samples, and the NucliSens HIV-1 QT and NucliSens Esay Q HIV-1 v1.1 and Versant HIV-1 RNA 3.0.bDNA reagents can also use heparin as an anticoagulant. If heparin-treated plasma samples must be used for RT-PCR amplification, heparin must be digested with heparinase directly in the sample.
2.2.3 Real-time fluorescent quantitative PCR technology
Real-time fluorescent quantitative PCR technology, is a method of adding fluorescent groups to the PCR reaction system, using fluorescent signal accumulation to monitor the entire PCR process in real time, and finally quantifying the unknown templates by a standard curve. There is a linear relationship between the number of cycles Ct value that the fluorescent signal in each reaction tube undergoes to reach a set domain value and the logarithm of the starting copy number of that template. (A standard curve can be made using a standard with a known starting copy number, where the horizontal coordinate represents the logarithm of the starting copy number and the vertical coordinate represents the Ct value). Therefore, by obtaining the Ct value of an unknown sample, the starting copy number of that sample can be calculated from the standard curve.
The experimental detection process of real-time fluorescence 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, detection of PCR products using fluorescence-labeled oligonucleotide probes. The principle of the assay is based on a fluorescence signal growth curve correlated with the number of cycles. (3) RT-PCR reaction, where viral RNA is reverse transcribed into cDNA using reverse transcriptase and then amplified by DNA polymerase for specific fragments. (4) Detection of amplification products, based on the detection threshold setting, fluorescence signal can be detected at low cycle number when the viral load is high, and fluorescence can be detected at high cycle number when the viral load is low. The cycle number is linearly related to the sample load. A standard curve of cycle number versus load can be made using standards to quantify the sample load. Using pooled nucleic acid amplification detection techniques and methods, window stage infections can be detected in a timely manner by performing pooled nucleic acid testing on samples that are highly suspected to be infected in the population and are antibody negative, utilizing the high sensitivity of nucleic acid detection methods. This method is more cost-effective than nucleic acid testing of single samples.
3.1 Sample pooling procedure
3.1.1 Based on the volume of pre-processed samples, calculate the number of pre-formed primary and secondary pools, and record the primary and secondary pools and the corresponding original sample numbers on the registration form.
3.1.2 Aspirate 130mL of sample and transfer to a centrifuge tube labeled Secondary Collection; 10 samples form a 1300mL Secondary Collection sample, which is fully vortexed and shaken to mix.
3.1.3 Aspirate 210mL of sample from each of the five secondary collection tubes into a centrifuge tube labeled Primary Collection; 10 samples form a Primary Collection consisting of 50 samples in a volume of 1050mL; vortex and shake well to mix.
3.1.4 Aspirate 500mL of pooled sample from each primary and secondary collection tube, dispense into another correspondingly labeled centrifuge tube, and assay with the Ultra Sensitive Nucleic Acid Detection Reagent.
3.1.5 Preparation of negative pooled external QCs: using 50 HIV antibody and nucleic acid negative samples, follow the steps above, respectively, to pool into 5 negative secondary pooled external QCs and 1 primary pooled external QC.
3.1.6 Preparation of Positive Pooled External QCs: From 9 HIV antibody and nucleic acid negative samples and one positive sample containing at least 10c/ml of HIV RNA, pipette 130mL into a centrifuge tube to form a 1,300mL Positive Secondary Pooled External QC. Then, from the four prepared negative secondary pooled external QCs and the above positive secondary pooled external QCs, respectively, 210 mL was pipetted into a centrifuge tube labeled as a primary positive pooled external QC to form a primary positive pooled external QC.
3.1.7 The primary and secondary negative and positive pooled external QCs are used for each round of RT-PCR for primary and secondary pooled samples, respectively.
3.2 Detection and breakdown routes for pooled samples
The use of commercialized nucleic acid detection reagents should be in strict accordance with the reagent instructions. Detection is performed according to the protocol of each commercialized nucleic acid reagent pooled PCR, and the number of pooled samples is operated according to each reagent protocol. The method is briefly described as follows:
3.2.1 The first level of pooled samples is tested by HIV RNA RT-PCR ultrasensitive detection, and the positively reacted first level of pooled samples proceeds to the next testing step.
3.2.2 All secondary pooled samples that comprise a positive primary pool are tested with the HIV RNA RT-PCR Ultrasensitive Assay, and positively reactive secondary pooled samples proceed to the next testing step.
3.2.3 Determine the nucleic acid-positive individual samples by testing 10 individual samples from all positive secondary pooled samples with the standard HIV RNA RT-PCR assay. HIV nucleic acid (DNA or RNA) testing can be used for early diagnosis of HIV infection in infants and children born to HIV-infected mothers up to 18 months after birth. Although the sensitivity of HIV RNA testing is higher early in the course of infection (within the first month of life), HIV DNA testing is not affected by interference with early diagnosis by maternal perinatal antiretroviral therapy and antiretroviral medications in human milk, or by prophylactic antiretroviral therapy in infants and young children. In addition, the use of umbilical cord blood for HIV nucleic acid testing is not recommended, given the contamination of the mother's blood. See Chapter 2 (HIV Antibody Testing) for antibody testing procedures and judgment of results in infants and young children.
4.1 Scope of application
Infants and young children born to HIV-infected mothers who are less than 18 months of age; infants and young children who are less than 18 months of age, whose mothers' HIV infection status is unknown, and whose children present with HIV-related clinical manifestations and are clinically suspected of HIV infection.
4.2 Testing Procedures and Reporting of Results
4.2.1 The first blood sample (blood samples can be prepared as DBS or EDTA anticoagulated whole blood) was collected from infants at 6 weeks (42 days) after birth and sent for testing.
4.2.2 If the first blood sample is positive, a second blood sample is collected as soon as possible for testing. If both blood samples are positive, report "Positive test result for early diagnosis of HIV infection in infants" and diagnose HIV infection in children. The HIV-infected child will be followed up and monitored in a timely manner, referred to a pediatric antiretroviral treatment provider, and provided with service measures such as prevention of opportunistic infections; if the second blood sample is negative, wait until the infant reaches 3 months of age to collect another blood sample for testing. If the first blood sample is negative, continue to provide child care and follow-up services, and wait until the infant reaches 3 months to collect blood samples for testing.
4.2.3 If the infant is tested negative again at 3 months of age, report "Negative results of early diagnosis of HIV infection in infants", and continue to provide child health care and follow-up services according to the treatment of uninfected children; when the child reaches the age of 12 months, report "Negative results of early diagnosis of HIV infection in infants" according to the "Procedures for testing of HIV antibodies in HIV-infected mothers and children". When the child reaches 12 months of age, according to the "HIV Antibody Testing Procedure for Children Born to HIV-Infected Mothers" (Figure 4), HIV antibody testing will be started to finally determine the child's infection status. If the infant tests positive again at 3 months of age, a second blood sample is collected as soon as possible. If the third blood sample is positive, report "Infant HIV infection early diagnosis test result is positive"; if the third blood sample is negative, report "Infant HIV infection early diagnosis test result is negative"; respectively, follow the above procedures to provide appropriate services. The service is provided accordingly.
4.2.4 Breastfed children who test negative for "Early Diagnosis of HIV Infection in Infants" at different times should have their blood collected again for qualitative nucleic acid testing for early diagnosis at 6 weeks and 3 months (or as soon as possible if they test positive at 6 weeks) after they stop breastfeeding completely. Antibody testing can be done directly after the child reaches 18 months of age.
4.2.5 If the infant is 3 months of age but less than 12 months at the time of the first blood collection, two blood samples should be collected as soon as possible at different times; both blood samples should be sent for testing at the same time, and the process should be followed as described above. If the child is 12 months old at the time of the first blood sample, the HIV antibody test should first be performed according to the "HIV Antibody Testing Procedure for Children Born to HIV-Infected Mothers" (Figure 4). If the results of two HIV antibody test reagents of different principles or manufacturers are negative, then the child is excluded from infection; if the results of the HIV antibody test reagents are positive and the child's infection status cannot be determined by the antibody test, then two blood samples can be collected at different times, and the "Early Diagnosis of HIV Infection in Infants" test can be performed in accordance with the aforementioned procedure. Early diagnosis of HIV infection in infants". If the child is 18 months of age or older at the time of the first blood collection, the HIV antibody test should be performed according to the HIV antibody test procedure (Figure 1), and the "Early Diagnosis of HIV Infection in Infants" test is not required.