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High-sensitivity detection method for human cytomegalovirus nucleic acid

A cytomegalovirus, high-sensitivity technology, applied in the direction of microbiological-based methods, biochemical equipment and methods, microbiological determination/testing, etc., can solve the problems of large impact on specimen storage time, sensitivity to be improved, and clinical application difficulties

Inactive Publication Date: 2013-06-19
程宁 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Among the existing detection methods, virus culture and isolation is the most accurate method, but it takes a long time, and the result is greatly affected by the storage time of the specimen, which is difficult in clinical application; the electron microscope is fast and accurate, and is generally suitable for laboratory research; It is widely used in HCMV research and diagnosis, but the detection sensitivity needs to be improved; while nucleic acid diagnosis has good sensitivity and specificity, which is the direction of application development

Method used

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  • High-sensitivity detection method for human cytomegalovirus nucleic acid
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  • High-sensitivity detection method for human cytomegalovirus nucleic acid

Examples

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example 1

[0085] When detecting human cytomegalovirus nucleic acid, the first round of PCR reaction system is: 10× amplification buffer 5ul, dNTPmix (10mmol) 1ul, upstream and downstream primers (10pmol / ul) each 1.5ul, viral template DNA 1ul, Taq DNA polymerization Enzyme (5U / ul) 0.5ul, MgCl2 (25mmol) 3ul, sterilized double distilled water to supplement 36.5ul.

[0086] The first round of PCR reaction conditions for detection of human cytomegalovirus nucleic acid is: pre-denaturation at 91°C for 5 minutes, cycle at 91°C for 30 seconds, 56°C for 1 minute, and 72°C for 1 minute, a total of 35 cycles, and extension at 72°C for 10 minutes.

[0087] When detecting human cytomegalovirus nucleic acid, its second-round PCR reaction system is: 10 × amplification buffer 5ul, dNTPmix (10mmol) 1ul, upstream and downstream primers (10pmol / ul) each 1.5ul, the first round of PCR amplification virus Nucleic acid product template DNA 5ul, Taq DNA polymerase (5U / ul) 0.5ul, MgCl2 (25mmol) 3ul, sterilized ...

example 2

[0091] When detecting human cytomegalovirus nucleic acid, the first-round PCR reaction system is: 10× amplification buffer 3ul, dNTPmix (10mmol) 1ul, upstream and downstream primers (10pmol / ul) each 1ul, viral template DNA 2ul, Taq DNA polymerization Enzyme (5U / ul) 1.0ul, MgCl2 (25mmol) 3ul, sterilized double distilled water to make up 50ul.

[0092] The first round of PCR reaction conditions for detection of human cytomegalovirus nucleic acid is: pre-denaturation at 90°C for 5 minutes, cycle at 88°C for 30 seconds, 52°C for 1 minute, and 68°C for 1 minute, a total of 35 cycles, and extension at 70°C for 10 minutes.

[0093] When detecting human cytomegalovirus nucleic acid, its second-round PCR reaction system is: 10× amplification buffer 3ul, dNTPmix (10mmol) 1ul, upstream and downstream primers (10pmol / ul) each 1ul, the first round of PCR amplifies viral nucleic acid Product template DNA 3ul, Taq DNA polymerase (5U / ul) 1.0ul, MgCl2 (25mmol) 3ul, sterilized double distilled ...

example 3

[0097] When detecting human cytomegalovirus nucleic acid, the first-round PCR reaction system is: 10× amplification buffer 3u1, dNTPmix (10mmol) 1ul, upstream and downstream primers (10pmol / ul) each 2ul, viral template DNA 2ul, Taq DNA polymerization Enzyme (5U / ul) 0.6ul, MgCl2 (25mmol) 3ul, sterilized double distilled water to make up 50ul.

[0098] The first round of PCR reaction conditions for detection of human cytomegalovirus nucleic acid was: pre-denaturation at 90°C for 5 minutes, cycle at 88°C for 30 seconds, 52°C for 1 minute, and 68°C for 1 minute for a total of 35 cycles, and extension at 71°C for 10 minutes.

[0099] When detecting human cytomegalovirus nucleic acid, its second-round PCR reaction system is: 10 × amplification buffer 5ul, dNTPmix (10mmol) 3ul, upstream and downstream primers (10pmol / ul) each 2ul, the first round of PCR amplification of viral nucleic acid Product template DNA 3ul, Taq DNA polymerase (5U / ul) 0.1ul, MgCl2 (25mmol) 1ul, sterilized doubl...

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Abstract

The invention relates to a detection and test method for cytomegalovirus nucleic acid. The method extracts a pathogenic DNA template, and uses pathogen specific lateral upstream and downstream primers for a first round of PCR amplification; the amplified product in the first round is used as a template to conduct a second round of PCR amplification by using pathogen specific inner upstream and downstream primers. A nested PCR method for detection of HCMV virus infection has sensitivity, specificity and authenticity superior to those of an ELISA method and a fluorescent quantitative PCR, so as to provide a high-sensitivity method for clinical practice.

Description

technical field [0001] The invention relates to a detection test method of cytomegalovirus nucleic acid, which can be applied in clinical examination and related research. Background technique [0002] Human cytomegalovirus (Human Cytomegalovirus, HCMV) is a DNA virus of the genus β in the family Herpesviridae, and natural infection is common in normal people. In the United States, the annual serum HCMV antibody positive conversion rate of children aged 3 to 10 is 3% to 6.2%; in the UK, the positive rate of HCMV antibody in young people is 10% to 15%, and it is 40% to 60% in upper-middle-class adults , up to 80% in the lower class of people; in developing countries, the antibody-positive rate of children under 3 years old reaches 80%, and almost 100% in the adult population. According to surveys, 70-90% of women of childbearing age in my country have had HCMV infection, 4.3-19.59% of pregnant women have HCMV infection during pregnancy, and the intrauterine transmission rate...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 程宁白亚娜
Owner 程宁
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