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Nucleic acid sequence combination for detecting type 4 herpes virus and application thereof

A nucleic acid sequence, herpes virus technology, which is applied in the directions of microorganism-based methods, recombinant DNA technology, and microorganism determination/inspection. Good specificity

Active Publication Date: 2020-05-05
宁波京慈生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Regarding the detection of EBV DNA, various PCR detection methods currently used are qualitative or quantitative by detecting specific gene fragments expressed during the incubation period, mainly including 2 kinds of EBV-encoded small RNAs (EBV-encoded small RNAs, EBERs), 6 kinds of Nuclear antigen (EBV nuclear antigen, EBNA) family and leading protein (leader protein, EBNA-LP), latent membrane protein (latent membrane protein, LMP), and Bam HI-A right transcripts (Bam HI-Arightward transcripts, BARTs ), due to the differences in the expression of EBERs and BARTs encoded by EBV in tumor patients and non-tumor patients, the real-time-PCR method for EBERs and BARTs is not suitable for the detection of latent non-tumor patients
At the same time, due to the differences in experimental design, PCR equipment and methods used in PCR research, there are obvious differences between experimental data.

Method used

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  • Nucleic acid sequence combination for detecting type 4 herpes virus and application thereof
  • Nucleic acid sequence combination for detecting type 4 herpes virus and application thereof
  • Nucleic acid sequence combination for detecting type 4 herpes virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The nucleic acid combination for detecting type 4 herpes virus comprises a pair of primers and a probe, the primer pair and probe are designed according to the unique coding EBV leading protein EBNA-LP of type 4 herpes virus.

[0063] Among them, the upstream primer is:

[0064] 5'-GGAGAGCTACTAAGAAGGCA-3' (SEQ ID NO.1)

[0065] Downstream primers are:

[0066] 5'-GGACACGTTACTTACCCCCTGAA-3' (SEQ ID NO.2)

[0067] The probes are:

[0068] FAM 5'-GAAGAGGAGGTGGTAAGC-3'HBQ1 (SEQ ID NO.3)

[0069] In this embodiment, the fluorescent reporting groups labeled at the 5' end of the probe are all FAM, and the quenching groups labeled at the 3' end are all HBQ1. It should be noted that, in other embodiments, the fluorescent reporting group is also It can be VIC or HEX, and the quenching group can be TAMRA.

[0070] For the detection of herpes virus type 4, the steps are as follows:

[0071] 1. Prepare the sample to be tested and establish a positive quality control: use the v...

Embodiment 2

[0079] In this example, the nucleic acid combination provided in Example 1 was used to verify the concentration ratio of primers and probes for real-time fluorescent quantitative PCR amplification of herpesvirus type 4.

[0080] According to the method provided in Example 1 (the annealing temperature is 55° C.), the real-time fluorescent PCR amplification experiment was carried out in the following primer probes with different concentration ratios.

[0081] The concentration ratio experiments of primers and probes are shown in Table 1 below.

[0082] Table 1 Primer Probe Concentration Ratio

[0083]

[0084] When the primer concentration in the reaction system is 600nM and the probe concentration is 400nM, the Ct value is small and the curve presents a smooth s-type, see figure 2 .

Embodiment 3

[0086] In this example, the nucleic acid combination provided in Example 1 was used to verify the Taq DNA polymerase for real-time fluorescent quantitative PCR amplification of herpes virus type 4.

[0087] This embodiment adopts the method in the first embodiment (annealing temperature is 55 ℃) to carry out PCR amplification under the concentration of Taq DNA polymerase of 0.25U, 0.5U, 1.0U, 1.5U respectively, compares the Taq DNA in different concentration The amplification efficiency of PCR under the polymerase, the result is as follows image 3 shown.

[0088] Depend on image 3 It can be seen that when the concentration of Taq DNA polymerase is 1-1.5U, the amplification efficiency of PCR is better.

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Abstract

The invention relates to the field of virus molecule detection, specifically to a nucleic acid sequence composition for detecting herpes virus IV and an application thereof. The nucleic acid sequencecomposition for detecting the herpes virus IV comprises the following primer pair and probes; the primer pair is shown in SEQ ID NO: 1 and SEQ ID NO: 2; a nucleic acid sequence of the probe is shown in SEQ ID NO: 3. The nucleic acid sequence composition for detecting the herpes virus IV, provided by the invention, comprises primers and the probe corresponding to the primers; the nucleic acid sequence composition for detecting the herpes virus IV, provided by the invention, is designed for specific genes EBNA-LP of the herpes virus IV and is used for detecting the herpes virus IV by combining areal-time PCR technology; the nucleic acid sequence composition for detecting the herpes virus IV, provided by the invention, has the advantages of high sensitivity and good specificity.

Description

technical field [0001] The invention relates to the field of virus molecular detection, in particular, to a combination of nucleic acid sequences for detecting type 4 herpes virus and its application. Background technique [0002] Epstein-Barr Virus (EBV) belongs to the Herpesviridae Gammaherpesviridae subfamily and is an important tumor-associated virus, which is related to the occurrence of various human lymphoid and epithelial cell tumors, such as Burkitt's lymphoma (Burkitt's lymphoma) s lymphoma, BL), Hodgkin's lymphoma, lymphocytoma in immunocompromised patients, nasopharyngeal carcinoma (NPC) and gastric cancer. EBV infection is very common, especially in childhood, and can be carried for life after infection. [0003] At present, clinical diagnosis of EBV infection mainly includes virus isolation and culture, serological detection, gene chip technology and PCR technology. The detection period of virus isolation and culture is long, and the detection rate of serolog...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/705C12Q2531/113C12Q2563/107
Inventor 邓铃何晓奕解庆华唐梦灵张雨
Owner 宁波京慈生物技术有限公司
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