The HIV genome consists of two single-stranded RNA, each RNA genome is about 9.7k, with a cap structure (m7g 5 PP 5'GMP NP) at the 5' end and a poly(A) tail at the 3' end. The main gene structure and combination form of HIV are the same as other retroviruses, and they are all composed of 5'-terminal repetitive sequence (LTR), structural protein coding region (gag), protease coding region (pro), protein coding region with various enzyme activities (pol), outer membrane protein (env) and 3'-terminal long repetitive sequence LTR region.
Nucleic acid was extracted by magnetic beads.
Principle of magnetic bead extraction;
Magnetic beads are marked with substances that specifically adsorb nucleic acids, and viral nucleic acids are extracted from the lysate by magnetic separation technology.
2) Step:
Cracking-adsorption-washing-washing-washing-elution
3) Advantages:
A. Magnetic bead extraction has high specificity, little influence by sample factors and high sensitivity.
B. Easy to realize automation
4) Disadvantages:
A. The cost is high, and certain instruments are needed (semi-automatic, fully automatic).
B.there's too much manual work.
Nested polymerase chain reaction (nPCR) was used for detection.
According to HIV- 1 gag.pol and env gene sequences, nesled polymerase chain reaction (nPCR) primers were designed. The lysate of peripheral blood mononuclear cells was amplified by external primers, and its products were amplified by internal primers. The results were directly determined by gel electrophoresis. Sensitivity of nPCR. It is 100 ~ 1000 times higher than conventional PCR, and can detect 0.5fg plasmid DNA and 50μ 1HIV- 1 HIV gene in infected peripheral blood samples.
According to professional literature, 63 HIV- 1 infected people were all positive by this method, while 6 1 healthy people and suspicious people were all negative. The latter ruled out HIV- 1 infection by antibody tracking.
Therefore, nPCR is a highly sensitive, specific and simple HIV- 1 gene detection technology. It plays an important role in early diagnosis and detection of peripheral blood virus content, and has a broader application prospect.
Advantages of HIV nucleic acid detection
It usually takes 2- 12 weeks for human to be infected with HIV, and it takes about 42 days to detect HIV antibody in blood on average. During this period, conventional detection technology can not detect HIV virus at all, which is usually called "HIV window period". The existence of "window period" also explains why some patients will be infected with AIDS after receiving blood transfusion in regular hospitals.
In the process of infection, viral RNA first appears, which can be detected by PCR, then IgM antibody is detected by double antigen sandwich method, and IgG antibody is detected by indirect method.
At present, the detection methods used by CDC and test paper in all provinces and cities are to detect antibody antigens, which cannot avoid the interference of "HIV window period" factors. Therefore, in the HIV window period, there are major defects in the conventional detection technology.