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Primer set and application thereof in amplifying SIV/SHIV (simian immunodeficiency virus/simian human immunodeficiency virus) genome, and kit

A primer amplification and genome technology, applied in the field of molecular biology, can solve problems such as sequence analysis problems, sequence selection and limited amplified sequences, recombination, etc., and achieve good sensitivity results

Active Publication Date: 2016-06-01
INST OF LAB ANIMAL SCI CHINESE ACAD OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the genome sequence length of SIV virus or SHIV virus is relatively long, about 10,000 bp, ordinary PCR technology often has the problems of recombination, sequence selection and limited amplified sequence, which makes the fragments obtained by amplification produce certain Mutations reduce the accuracy and cause trouble for subsequent sequence analysis

Method used

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  • Primer set and application thereof in amplifying SIV/SHIV (simian immunodeficiency virus/simian human immunodeficiency virus) genome, and kit
  • Primer set and application thereof in amplifying SIV/SHIV (simian immunodeficiency virus/simian human immunodeficiency virus) genome, and kit
  • Primer set and application thereof in amplifying SIV/SHIV (simian immunodeficiency virus/simian human immunodeficiency virus) genome, and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Using the SIVmac239 plasmid as a template, the primers listed in Table 1 were amplified.

[0067] Table 1 Primer Sequence

[0068]

upstream

downstream

Amplified fragment length

primer pair 1

SEQ ID NO:1

SEQ ID NO:2

5728bp

primer pair 2

SEQ ID NO:9

SEQ ID NO:10

5398bp

Primer pair 3

SEQ ID NO:9

SEQ ID NO: 11

5381bp

primer pair 4

SEQ ID NO:3

SEQ ID NO:4

5103bp

primer pair 5

SEQ ID NO:12

SEQ ID NO: 13

5022bp

Primer pair 6

SEQ ID NO: 14

SEQ ID NO:6

4811bp

primer pair 7

SEQ ID NO:5

SEQ ID NO:6

4842bp

Primer pair 8

SEQ ID NO:7

SEQ ID NO:8

4766bp

[0069] The amplification system is:

[0070]

[0071] The amplification procedure is:

[0072]

[0073]

[0074] The amplified product was subjected to 1.2% TAE agarose gel electrophoresis, and the results were as follows: figure 1 , wherein, ...

Embodiment 2

[0076] The primers in Table 2 were used to amplify by means of nested PCR. The first round of amplification used the culture supernatant of SIVmac239 as a template, and the second round of amplification used the amplification product of the first round as a template. Experimental setup Two experimental groups were used to amplify the 3' half-length and 5' half-length of the genome respectively. Another 10 comparison groups were set up.

[0077] Table 2 Primer Sequence

[0078]

[0079]

[0080] The amplification system is:

[0081]

[0082] The amplification procedure is:

[0083]

[0084] After two rounds of PCR amplification in each group of experiments, the products were subjected to 1.2% TAE agarose gel electrophoresis, and the results were as follows: Figure 2-a with Figure 2-b . It can be seen from the figure that the primer pair provided by experimental group 1 is most suitable for amplifying the 5' end of the SIV genome, while the primer pair provide...

Embodiment 3

[0087] The primers in Table 2 in Example 2 were used to amplify by means of nested PCR. The first round of amplification used the SIVmac239 plasmid as a template, and the second round of amplification used the amplification product of the first round as a template. Experimental setup Two experimental groups were used to amplify the 3' half-length and 5' half-length of the genome respectively. Another 10 comparison groups were set up.

[0088] The amplification system is:

[0089]

[0090] The amplification procedure is:

[0091]

[0092] After two rounds of PCR amplification in each group of experiments, the products were subjected to 1.2% TAE agarose gel electrophoresis, and the results were as follows: Figure 3-a with Figure 3-b . It can be seen from the figure that the primer pair provided by experimental group 1 is most suitable for amplifying the 5' end of the SIV genome, while the primer pair provided by experimental group 2 is most suitable for amplifying th...

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Abstract

The invention relates to the field of molecular biology, particularly a primer set and application thereof in amplifying SIV / SHIV (simian immunodeficiency virus / simian human immunodeficiency virus) genome, and a kit. The primer set provided by the invention can be used for specific, stable and accurate amplification to obtain the target sequence. The agarose gel electrophoresis detection indicates that no non-specific strip is generated, and no trailing or dispersion is generated. Compared with the SIVmac239 whole genome sequence registered in Genbank after sequencing, the matching degree is 100%, and no base replacement or deletion (gap) is detected. The experiment indicates that the primer set has favorable sensitivity for the SIV / SHIV genome and can have favorable detection effects when the virus copy number is 100.5-1.0, and thus, the analysis judges that the sensitivity difference is related to virus sequence variation degree.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a primer set and its application and kit in amplifying SIV / SHIV genome. Background technique [0002] AIDS, also known as acquired immunodeficiency syndrome (Acquired Immune Deficiency Syndrome, AIDS) is a class of infectious diseases with high mortality caused by human immunodeficiency virus (Humanimmunodeficiencyvirus, HIV), which affects the whole world, especially in Asia and Africa. The life, health and safety of people in developing countries are greatly threatened. One of the most unique features of HIV is the high variability of the genome. Based on genetic variation, HIV-1 that is prevalent in the world is divided into four groups, M group, O group, N group and P group, and M group can be divided into A~J10 subtypes, and the genetic dispersion rate of each subtype It is 20%-35%. At the same time, there are dozens of circulating recombinant types. Viral genome gene ana...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10
CPCC12N15/1003C12N15/11C12Q2527/101C12Q2531/113C12Q2549/119
Inventor 王卫高峰秦川魏强
Owner INST OF LAB ANIMAL SCI CHINESE ACAD OF MEDICAL SCI
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