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Cytomegalovirus detecting method and kit thereof by nucleic acid isothermal amplication

A cytomegalovirus and detection kit technology is applied in the field of cytomegalovirus nucleic acid amplification and detection to achieve the effects of reasonable design, practical clinical significance and application value, and improved detection sensitivity and specificity

Inactive Publication Date: 2010-06-30
SHANGHAI FOSUN PHARMA (GROUP) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no public report on the application of NITAG technology to detect CMV mRNA. The detection can reflect the activation state of the virus in the biological host, which has practical clinical significance

Method used

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  • Cytomegalovirus detecting method and kit thereof by nucleic acid isothermal amplication
  • Cytomegalovirus detecting method and kit thereof by nucleic acid isothermal amplication

Examples

Experimental program
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Effect test

Embodiment 1

[0042] CMV mRNA NITAG Detection of CMV Cultures

[0043] The implementation of the present invention is exemplified below by introducing mRNANITAG detection of cytomegalovirus culture extracts.

[0044] RNA extraction : The sample is the CMVAD169 standard strain culture grown in human embryonic lung fibroblasts. After diluting with normal saline, take 250 μl and add 500 μl Trizol extraction reagent, repeatedly blow and mix with a pipette, leave at room temperature for 5 minutes, add 200 μl chloroform, Shake back and forth by hand for 15 seconds, place at room temperature for 5 minutes, centrifuge at 12,000 rpm at 4°C for 15 minutes, transfer the supernatant to a new centrifuge tube, add 200 μl of isopropanol, vortex for 5 seconds, and precipitate in an ice bath for 10 minutes, at 4°C Centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, add 500 μl of pre-cooled DEPC water to prepare 75% ethanol for washing, invert up and down 10 times, centrifuge at 10,000 rpm at...

Embodiment 2

[0047] NITAG Detection of CMV mRNA in Organ Transplant Patient Samples

[0048] Pretreatment method : The samples are anticoagulated whole blood of patients who have undergone clinical organ transplantation (kidney, liver and bone marrow) and are receiving immunosuppressive therapy. Take 1ml of each sample and wash it repeatedly with red blood cell lysate, separate white blood cells and resuspend in 1ml of normal saline , ready for RNA extraction.

[0049] RNA extraction : Take 250 μl of white blood cells separated above, add 500 μl of Trizol extraction reagent, pipette repeatedly to mix, leave at room temperature for 5 minutes, add 200 μl of chloroform, shake back and forth by hand for 15 seconds, leave at room temperature for 5 minutes, 4°C 12,000rpm Centrifuge for 15 minutes, transfer the supernatant to a new centrifuge tube, add 200 μl of isopropanol, vortex for 5 seconds, precipitate in an ice bath for 10 minutes, centrifuge at 12,000 rpm at 4°C for 10 minutes, discar...

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Abstract

The present invention relates to a cytomegalovirus (CMV) detecting method and a kit thereof by nucleic acid isothermal amplication. The detection includes the steps in sequence of unwinding the mRNA template of CMV under 60 to 70 DEG C and then cooling, adding with reverse transcriptase, ribonuclease H, RNA polymerase, performing the isothermal amplication under 37 to 42 DEG C conducted by primer to obtain RNA amplicon, then detecting the RNA amplicon by gold nanometer probe and capture probe, and obtain the detecting result through chromatography cross-chromogenic reaction. The mRNA is positive if chromogenic stripe appears on the cross film and the RNA is negative or else. The kit of the method can detect the mRNA of CMV quickly and accurately.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a cytomegalovirus nucleic acid amplification detection technology. Background technique [0002] Cytomegalovirus (CMV), as a DNA virus belonging to the family Herpesviridae, can infect the fetus through the placenta, causing premature birth, fetal growth retardation, neonatal malformation, jaundice, hepatosplenomegaly, hemolytic anemia, retinal choroid CMV infection is very common in immunocompromised patients such as AIDS, cancer, organ transplantation, etc. After infection with CMV, progressive interstitial pneumonia, hepatitis, encephalitis, pericardial Inflammation and disseminated CMV infection often threaten the life of patients and affect the survival of organ transplantation. [0003] CMV infection is very common in humans, and most of them have no clinical symptoms and are latent and will not be eliminated. Acquired infections include contact infections with uri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/76
Inventor 吴大治夏懿
Owner SHANGHAI FOSUN PHARMA (GROUP) CO LTD
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