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Method of detecting and quantifying cytomegalovirus

A cytomegalovirus, specific sequence technology, applied in biochemical equipment and methods, microbial measurement/inspection, fermentation, etc., can solve the problem of amplification, poor detection efficiency, hindering oligonucleotide binding, primers and detection The probe does not know the problem, to achieve the effect of high sensitivity and rapid sensitivity

Inactive Publication Date: 2007-01-03
TOSOH CORP
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Problems solved by technology

[0009] However, primers and probes for amplification of CMV-derived mRNA suitable for the method of Patent Document 4 are not yet known.
This is because the method of Patent Document 4 is considered to carry out the amplification and detection of mRNA at a certain temperature, and the high-order structure of the target mRNA hinders the binding of oligonucleotides, so the efficiency of amplification and detection deteriorates.

Method used

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  • Method of detecting and quantifying cytomegalovirus

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Embodiment 1

[0042] Preparation of intercalating fluorochrome-labeled oligonucleotide probes.

[0043] The phosphorus atom between the 4th base (G) and the 5th base (C) from the 5' end of the sequence shown in SEQ ID NO: 9 is labeled with the well-known pigment oxazolone as an intercalating fluorescent pigment, and  Azoflavin-labeled oligonucleotide probe (SEQ ID NO: 9) (see Ishiguro, T (1996) Nucleic Acids Res, 24 (24) 4992-4997).

Embodiment 2

[0045] Using the combination of oligonucleotide primers invented by the present application, various initial copy numbers of β2.7 RNA are detected.

[0046] (1) Preparation of β2.7RNA

[0047] The so-called β2.7 RNA is a double-stranded DNA of 2154 bases in the base sequence of the β2.7 gene derived from CMV (accession number of the National Center for Biotechnology Information: 2471 bases from 184889 to 187359 of X17403) After cloning, synthesized and purified RNA is used as a template by in vitro transcription.

[0048] β2.7RNA (2154mer) was used as a sample, quantified according to the UV absorption at 260nm, and diluted with RNA diluent (10mM Tris-HCl (pH8.0), 0.1mM EDTA, 0.5U / μl ribonuclease inhibitor, 5.0mMDTT) into 1.0×10 6 copies / 5μl~30 copies / 5μl. Only RNA dilutions were used in the control experimental area (nega).

[0049] (2) 20.0 µl of a reaction solution having the following composition was dispensed into a 0.5 ml-capacity PCR tube (thin-walled reaction tube ...

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Abstract

Provided are detection and assay of CMV that can rapidly amplify and detect the beta 2.7 gene originating from CMV at a certain temperature through a one-step operation. The mRNA of beta 2.7 gene originating from CMV is amplified by utilizing the combination of specifically amplifiable oligonucleotide primers and the RNA amplification step comprising these combinations of oligonucleotide primers is measured and detected by using an oligonucleotide probe that is marked with an intercalated fluorescent dye, to settle above topic.

Description

technical field [0001] The invention relates to a method for detecting and quantifying CMV by using a specific nucleic acid sequence. Background technique [0002] Cytomegalovirus (CMV) is an icosahedral DNA virus belonging to the β-human herpesviridae family, and 90% of Japanese adults are infected with the virus. Infection is usually just after birth, but since most infections are asymptomatic, no special symptoms appear. Acquired infection includes contact infection with urine, saliva, breast milk and semen as the source of infection, and nosocomial infection caused by blood transfusion or organ transplantation. The characteristic of CMV is that it stays latent in the living body throughout its life, and can be reactivated (reactivated) from a latent infection state due to physiological changes of the host, and can repeatedly and periodically develop disease. In malignant tumors, AIDS patients, and organ transplant patients, the immunodeficiency state becomes the cause ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12Q1/68C12Q1/70
CPCC12Q1/705C12Q1/6844C12Q2563/173
Inventor 大仲悟林俊典
Owner TOSOH CORP
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