A detection kit for accurately quantifying HIV DNA and its detection method

A technology for detection kits and detection methods, which can be used in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc. Positive results, improved sensitivity, improved specificity

Inactive Publication Date: 2019-10-08
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Compared with real-time quantitative PCR technology, the advantage of digital PCR technology is that it can quantify a large number of DNA templates, but the disadvantage is that chips are required every time, and the price is relatively expensive.
In addition, because the nested PCR uses the second pair of primers to amplify the first round of PCR products, in terms of qualitative aspects, the specificity and sensitivity of the amplification will be greatly improved, but because the nested PCR primers are combined A second round of amplification within a PCR product, which cannot be quantified accurately

Method used

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  • A detection kit for accurately quantifying HIV DNA and its detection method
  • A detection kit for accurately quantifying HIV DNA and its detection method
  • A detection kit for accurately quantifying HIV DNA and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Establishment of nested PCR method

[0035] 1. DNA preparation

[0036] The standard product cell line-Jurket cell line according to 10 6 、10 5 、10 4 、10 3 、10 2 After doubling dilution, extract the DNA, and use 60 μl of the system to dissolve the DNA in the final step of collecting the DNA. The copy numbers of HIV DNA contained in 1 μl are 17000, 1700, 170, 17, and 1.7 respectively as standards;

[0037] 2. PCR amplification

[0038] 2.1 Primer design

[0039] In nested PCR, two sets of primers (A and B) were designed (as shown in Table 1), and each set consisted of two pairs of primers, outer (outer coat) and inner (inner set), in order to improve the sensitivity and specificity of the experiment. The concentration is 0.4 μmol / L.

[0040] Table 1 Nested PCR primer sequences

[0041]

[0042]

[0043] 2.2 Reaction system and reaction conditions

[0044] The reaction system was prepared according to Table 2. According to the reaction conditions...

Embodiment 2

[0053] Example 2 Establishment of real-time fluorescent quantitative PCR method

[0054] 1, DNA preparation Same as Example 1

[0055] 2. Primer and probe design

[0056] A total of five pairs of primers P1-P5 were designed, respectively corresponding to HIV DNA, CCR5 and three HIV subtypes AE, BC, and B (Table 5). Using the characteristics that each cell contains two copies of CCR5, the number of cells is quantified by amplifying CCR5, thereby calculating the number of cells per 10 6 The average amount of HIV DNA contained in a cell.

[0057] Table 5 Real-time fluorescent quantitative PCR primers and probe sequences

[0058]

[0059]

[0060] Remark:

[0061] ①P5 upstream primer is the same as P2 upstream primer

[0062] ② Probe 108 uses FAM fluorescence, probe 136 uses VIC fluorescence

[0063] ③P1 primers use 136 probes, and P2, P3, P4, and P5 primers all use 108 probes

[0064] ④The underlined part of the 136 probe is the hairpin structure.

[0065] 3. Perfor...

Embodiment 3

[0080] The establishment of embodiment 3 Digital-PCR method

[0081] Prepare the reaction system according to Table 9, add 25X stabilizer at the end, and use RainDance's mature RainStrom TM Skin droplet patented technology for oil droplet preparation. PCR amplification was carried out after the oil droplets were prepared, and the reaction conditions were shown in Table 7. After PCR amplification, the instrument was used to detect the fluorescence count. Digital PCR collects the fluorescent signal of each reaction unit after the amplification is completed. A fluorescent signal was recorded as 1, and no fluorescent signal was recorded as 0.

[0082] Since digital PCR calculates the copy number of the target sequence through endpoint detection, accurate absolute quantitative detection can be performed without using internal references and standard curves. In addition, since digital PCR uses endpoint detection, it does not depend on the Ct value, so digital PCR is greatly redu...

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Abstract

The invention discloses a detection method for accurately quantifying HIV DNA in cooperation with multiple PCRs. Particularly, a nested PCR, a real-time fluorescence quantification PCR and a Digital PCR are used in a combined mode to accurately quantify the HIV DNA; through combination of the three PCR methods, specific primers for all the subtypes of HIV are designed, and the aims of accurate quantification and cost saving of the HIV DNA are achieved; in addition, a detection kit for accurately quantifying the HIV DNA according to the detection method is developed, and the detection kit has broad application prospects in clinic.

Description

technical field [0001] The invention belongs to the field of molecular detection and relates to a detection kit for accurately quantifying HIV DNA, in particular to a detection kit for accurately quantifying HIV DNA combined with multiple PCR methods. Background technique [0002] At present, ART treatment can reduce HIV RNA to undetectable levels. Due to the existence of HIV virus reservoir, ART treatment cannot be stopped. HIV virus reservoir mainly exists in the form of HIV DNA, and the virus storage in the form of HIV DNA Reservoirs become an obstacle to HIV eradication. Studies have shown that the level of HIV DNA is closely related to the time of viral rebound after drug withdrawal. The lower the HIV DNA level, the longer it took for the virus to rebound after stopping the drug. Therefore, accurate quantification of HIV DNA is very important. Due to the very low amount of HIV DNA, it is very difficult to accurately quantify it. [0003] Currently, quantitative PCR ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
Inventor 焦艳梅徐若男王福生
Owner THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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