Nested PCR primers and method for detecting drug-resistant mutation of cytomegalovirus UL54 and UL97 genes

A cytomegalovirus, UL97-SR2 technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of not being able to be promoted on a large scale, time-consuming and labor-intensive equipment, and not being able to cover them all, and achieve high specificity High sensitivity, high practicability and strong effect

Inactive Publication Date: 2017-05-10
DONGGUAN EIGHTH PEOPLES HOSPITALDONGGUAN CHILDRENS HOSPITAL +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, traditional gene mutation diagnosis methods include restriction fragment length polymorphism analysis (RFLP), gene chip method, fluorescent quantitative PCR, etc. These methods have the disadvantages of time-consuming and labor-intensive or expensive equipment and consumables, which prevent them from being widely used in clinical practice. Scale promotion, limited application
At the same time, there are many drug-resistant mutation sites in cytomegalovirus (HCMV) UL54 and UL97 genes, and the above methods cannot cover all of these sites

Method used

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  • Nested PCR primers and method for detecting drug-resistant mutation of cytomegalovirus UL54 and UL97 genes
  • Nested PCR primers and method for detecting drug-resistant mutation of cytomegalovirus UL54 and UL97 genes
  • Nested PCR primers and method for detecting drug-resistant mutation of cytomegalovirus UL54 and UL97 genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] In this embodiment, a nested PCR primer for detecting the drug-resistant mutation of cytomegalovirus UL54 gene, the nested PCR primer includes the first round of PCR amplification primers and the second round of PCR amplification primers:

[0046] When detecting cytomegalovirus UL54,

[0047] Primers for the first round of PCR amplification are:

[0048] The first pair of primers: forward primer UL54-SF1 and reverse primer UL54-SR1;

[0049] The second round of PCR amplification primers is the second pair of primers and the third pair of primers:

[0050] The second pair of primers: forward primer UL54-SF2 and reverse primer UL54-SR2;

[0051] The third pair of primers: forward primer UL54-SF3 and reverse primer UL54-SR3;

[0052] In this example, the first pair of primers was used for the first round of amplification of the UL54 gene, including the forward primer UL54-SF1 (SEQ ID No.1) and the reverse primer UL54-SR1 (SEQ ID No.2); the second pair Primers were used...

Embodiment 2

[0073] In this embodiment, a nested PCR primer for detecting the drug-resistant mutation of cytomegalovirus UL97 gene, the nested PCR primer includes the first round of PCR amplification primers and the second round of PCR amplification primers:

[0074] When detecting cytomegalovirus UL97,

[0075] Primers for the first round of PCR amplification are:

[0076] The fourth pair of primers: forward primer UL97-SF1 and reverse primer UL97-SR1;

[0077] The primers for the second round of PCR amplification are:

[0078] The fifth pair of primers: forward primer UL97-SF2 and reverse primer UL97-SR2;

[0079] In this example, the fourth pair of primers is used for the first round of amplification of the UL97 gene, including the forward primer UL97-SF1 (SEQ ID No.7) and the reverse primer UL97-SR1 (SEQ ID No.8); the fifth pair The primers are used for the second round of amplification of UL97 gene, including forward primer UL97-SF2 (SEQ ID No.9) and reverse primer UL97-SR2 (SEQ ID...

Embodiment 3

[0099] The difference between this embodiment and embodiment 1 is:

[0100] In this embodiment, in the step (1), the volume of the first-round PCR reaction system is 25 μL. Specifically, the reaction system of the first round of PCR is: KOD FX 0.5U and dNTPs 0.4mM each, 2×PCR buffer for KOD FX 12.5 μL, 10 pmol / μL forward primer and reverse primer 0.75 μL each, and DNA to be detected 1 μL, make up to 25 μL with double distilled water. Wherein, the first-round PCR product is diluted 50 times to obtain the first-round PCR diluted product.

[0101] In this embodiment, in the step (2), the volume of the reaction system of the second round of PCR is 50 μL. Specifically, the reaction system for the second round of PCR was: KOD FX 1U and dNTPs 0.4 mM each, 2×PCR buffer for KOD FX 25 μL, 10 pmol / μL forward primer and reverse primer 0.75 μL each, and the first round of PCR dilution 1 μL of the product was made up to 50 μL with double distilled water.

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Abstract

The invention relates to the technical field of detection for virogene mutation, and in particular relates to nested PCR primers and a nested PCR method for detecting drug-resistant mutation of cytomegalovirus UL54 and UL97 genes. Five pairs of primers are adopted, for the UL54 gene, the primers for first round of PCR amplification are the first pair of primers, the primers for second round of PCR amplification are the second and third pairs of primers, and the sequences of the primers are shown as SEQ ID No.1-6; for the UL97 gene, the primers for first round of PCR amplification are the fourth pair of primers, the primers for second round of PCR amplification are the fifth pair of primers, and the sequences of the primers are shown as SEQ ID No.7-10. For the nested PCR primers provided by the invention, the nested PCR method is adopted, the PCR reaction system and reaction procedure are optimized, the amplification for the UL54 and UL97 genes has high sensitivity and high amplification specificity, the primers and the method can be used for analyzing the drug resistance gene mutation, the practicability is strong, and the demands for clinical promotion are satisfied.

Description

technical field [0001] The invention relates to the technical field of viral gene mutation detection, in particular to nested PCR primers and a method for detecting drug-resistant mutations of cytomegalovirus UL54 and UL97 genes. Background technique [0002] HCMV belongs to Herpesviridae, Betaherpesvirinae, Cytomegalovirus, and Human Herpesvirus Type 5. The HCMV genome is a linear double-stranded DNA molecule, about 230 kb in length, consisting of a long single sequence (UL) and a short single sequence (US). HCMV exists widely in nature, and natural infection is common in normal population. [0003] Human cytomegalovirus (HCMV) is an opportunistic pathogen in immunocompromised or immature populations, most infected persons are asymptomatic, but in congenitally infected children, or in immunosuppressed individuals such as organ transplants In patients with acquired immunodeficiency syndrome (AIDS), it is easy to activate infection, cause serious lesions, and endanger life....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/705C12Q1/6848C12Q1/6858C12Q2600/156C12Q2531/113C12Q2549/119
Inventor 黎四平马强陆小梅钟柏茂谢明玉彭琪饶春宝
Owner DONGGUAN EIGHTH PEOPLES HOSPITALDONGGUAN CHILDRENS HOSPITAL
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