The magnetic bead method is used for nucleic acid extraction.
1) Principle of magnetic bead extraction:
The magnetic beads are labeled with substances that specifically adsorb nucleic acids to specifically adsorb nucleic acids, and the viral nucleic acids are extracted from the lysate through magnetic separation technology. Extract it.
2) Steps:
Cracking - adsorption - washing - washing - washing - elution
3) Advantages:
a. Magnetic bead extraction has high specificity, is less affected by specimen factors, and has high sensitivity
b. Easy to automate
4) Disadvantages:
a. High cost , and requires certain instruments (semi-automatic, fully automatic).
b. Completely manual work, the workload is too large. Nested polymerase chain reaction (nPCR) primers were designed based on HIV-1 gag.pol and env gene sequences. Peripheral blood mononuclear cell lysate is first amplified with outer primers, and the product is amplified with inner primers, and the results are directly determined by gel electrophoresis. sensitivity of nPCR. It is 100 to 1000 times higher than conventional PCR and can detect HIV genes in 0.5fg plasmid DNA and 50μ1 peripheral blood samples of HIV-1 infected persons.
According to professional literature, 63 HIV-1-infected people tested using this method were all positive, while 61 healthy people and suspects were all negative. The latter were ruled out for HIV-1 through follow-up antibody testing. Infect.
It can be seen that nPCR is an HIV-1 gene detection technology with extremely high sensitivity, strong specificity, and simple and easy operation. It has already played an important role in early diagnosis and peripheral blood virus content detection, and has broader application prospects.
Advantages of testing
After a human being is infected with HIV, it generally takes 2-12 weeks, and an average of about 42 days, before HIV antibodies can be detected in the blood. During this period, conventional testing techniques cannot detect the HIV virus at all. This is often referred to as the "HIV window period." The existence of the "window period" also explains why some patients become infected with AIDS after receiving blood transfusions in regular hospitals.
During the infection process, the first thing that appears is viral RNA, which can be detected by PCR. Then IgM antibodies appear, which can be detected using the double-antigen sandwich method. Then, what appears is IgG antibodies can be detected using indirect methods.
The detection methods currently used by provincial and municipal disease control centers and test strips all detect antibody antigens, which cannot avoid the interference of the "HIV window period" factor. Therefore, conventional testing technology has major flaws during the HIV window period.