Primer group, detection method and kit for detecting drug resistance of cytomegalovirus

A cytomegalovirus, primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc., can solve the problems of unsuitable large-scale batch detection, poor detection specificity, low sensitivity, etc. The effect of high sex, strong specificity and short amplification time

Inactive Publication Date: 2020-06-26
倍科为(天津)生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The phenotype method is to let a certain amount of virus grow in the medium equipped with different concentrations of antiviral drugs through cell culture, and finally calculate the half-inhibitory concentration (IC50) of the drug, and divide the virus into sensitive and resistant according to the experimental results. Drug type, because the HCMV virus culture time generally takes 1 to 14 days, so this method is time-consuming, laborious, and not suitable for large-scale batch detection; the genotype detection method is to amplify the relevant drug resistance genes of HCMV clinical isolates. By adding and sequencing, it is judged whether the strain is a drug-resistant strain by comparing with the sequence of the sensitive gene. However, due to the large number of drug-resistant mutation sites in the UL97 gene, the existing genotype detection methods cannot cover all of these sites at all. And there is the defect of poor detection specificity and low sensitivity

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  • Primer group, detection method and kit for detecting drug resistance of cytomegalovirus
  • Primer group, detection method and kit for detecting drug resistance of cytomegalovirus
  • Primer group, detection method and kit for detecting drug resistance of cytomegalovirus

Examples

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Effect test

Embodiment 1

[0071] This example provides a nested PCR method using nested PCR primers to detect drug-resistant mutations in the cytomegalovirus UL97 gene.

[0072] This embodiment adopts the method of double nested PCR, wherein the sequence of the first pair of primers UL97-F1 and the first pair of primers UL97-R1 of the first nested PCR is shown in Table 1, and the first round of nested PCR amplification reaction The program is as follows: (1) 94°C, pre-denaturation for 3 min; (2) 94°C, denaturation for 30 s, 58.5°C for 35 s, 72°C for 1 min, a total of 35 cycles; (3) 72°C for 5 min.

[0073] Table 1 The first round of nested PCR amplification reaction program and primer list.

[0074]

[0075] The system of the first round of nested PCR reaction is shown in Table 2.

[0076] Table 2 The first round of nested PCR reaction system.

[0077]

[0078]

[0079] Template DNA: Take the patient's whole blood for nucleic acid extraction.

[0080] Wherein the sequence of the second pair...

Embodiment 2

[0140] This example provides a nested PCR method using nested PCR primers to detect drug-resistant mutations in the cytomegalovirus UL97 gene.

[0141] This embodiment adopts the method of one-fold nested PCR, wherein the sequences of the first pair of primers UL97-F1 and the first pair of primers UL97-R1 of nested PCR are the same as those in Example 1, and the first round of nested PCR amplification reaction procedure is as follows: (1) Pre-denaturation at 94°C for 3 min; (2) Denaturation at 94°C for 30 s, annealing at 58.5°C for 35 s, extension at 72°C for 1 min, a total of 35 cycles; (3) extension at 72°C for 5 min.

[0142] The system of the first round of nested PCR reaction was also the same as in Example 1.

[0143] The sequence of the second pair of primers UL97-F2 and the second pair of primers UL97-R2 in the second round of nested PCR is also the same as in Example 1, and the second round of nested PCR amplification reaction program is as follows: (1) 94 ° C, pre-de...

Embodiment 3

[0146] This example provides a nested PCR method using nested PCR primers to detect drug-resistant mutations in the cytomegalovirus UL97 gene.

[0147] This embodiment adopts a method of nested PCR, wherein the sequences of the third pair of primers UL97-F3 and the third pair of primers UL97-R3 in the first round of nested PCR are the same as those in Table 5 in Example 1, and the first round of nested PCR The PCR amplification reaction program was as follows: (1) 94°C, pre-denaturation for 3 min; (2) 94°C, denaturation for 30 s, 55°C for 45 s, 72°C for 45 s, a total of 25 cycles; (3) 72°C for 5 min.

[0148] The system of the first round of nested PCR reaction is the same as that in Table 6 in Example 1.

[0149] Template DNA: Take the patient's whole blood for nucleic acid extraction. Patient's sent specimen reference Image 6 shown.

[0150] The sequence of the fourth pair of primers UL97-F2 and the fourth pair of primers UL97-R2 in the second round of nested PCR is iden...

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Abstract

The invention discloses a primer group, a detection method and a kit for detecting drug resistance of cytomegalovirus. The primer group can highly specifically amplify a target fragment of a cytomegalovirus UL97 gene, can be used for analyzing drug resistance gene site mutation, and provides theoretical guidance for clinical use. The detection method provided by the invention is simple and accurate to operate and high in specificity.

Description

technical field [0001] The invention relates to the technical field of viral gene mutation detection, in particular to a primer set, detection method and kit for detecting cytomegalovirus drug resistance. Background technique [0002] Human cytomegalovirus (HCMV) is the largest member of the herpes virus family genome, which can encode more than 200 proteins. HCMV mainly infects humans, and there is no animal infection model. The cleavage, replication and proliferation of HCMV are slow and the cycle is long. In addition to the formation of nuclear inclusion bodies, HCMV has the characteristics of triggering the production of perinuclear and cytoplasmic inclusion bodies and the formation of giant cells, hence the name. [0003] HCMV infection is very common in the population, as high as 60% to 80% of the population in my country is positive for HCMV antibodies. 10% to 15% of children are infected with HCMV for the first time before the age of 5, and the proportion of infect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/701C12Q2549/119C12Q2537/165
Inventor 卓频
Owner 倍科为(天津)生物技术有限公司
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