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Primer sequences and quantitative determination kit used for simultaneously detecting human CMV and BK virus DNA

A quantitative detection and virus technology, applied in the direction of recombinant DNA technology, DNA / RNA fragments, microbial measurement / inspection, etc., to achieve the effect of multi-experimental information, simple composition, and reduced manpower and material resources

Inactive Publication Date: 2013-01-23
THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no other method to quantitatively detect the DNA of CMV and BKV at the same time

Method used

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  • Primer sequences and quantitative determination kit used for simultaneously detecting human CMV and BK virus DNA
  • Primer sequences and quantitative determination kit used for simultaneously detecting human CMV and BK virus DNA
  • Primer sequences and quantitative determination kit used for simultaneously detecting human CMV and BK virus DNA

Examples

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Embodiment 1

[0031] Example 2

Embodiment 2

[0033] Example 3

Embodiment 3

[0035] Reagent A: A 250 μL solution is composed of a mixture of primers and probes for CMV and BKV. The solution contains 5 μM SEQ ID NO.1, 5 μM SEQ ID NO.2, 5 μM SEQ ID NO.4, 5 μM SEQ ID NO.5 and 2 μM probe SEQ ID NO.3, 2 μM probe SEQ ID NO.6;

[0036] Reagent B: Consists of 250 μL buffer containing 1 mM dNTPs (including dATP, dCTP, dGTP, dTTP), 200 mM Tris-HCl, 200 mM KCl, 100 mM (NH 4 ) 2 SO 4 , 45 mM MgSO 4 ;

[0037] Reagent C: 5U / μL Taq DNA polymerase;

[0038] Reagent D: The prepared standard was serially diluted to reach 5E7 (5×10 7 copy / mL), 5E6, 5E5, 5E4, 5E3, used for standard curve formulation;

[0039] Reagent E: negative control (normal human DNA without CMV and BKV infection);

[0040] Reagent G: Positive control (using 5E4 standard).

[0041] Example 4

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Abstract

The invention discloses primer sequences and a quantitative determination kit used for simultaneously detecting human CMV and BK virus DNA. The invention discloses primers, fluorescent probes, and a real-time quantitative polymerase chain reaction (real-time PCR) kit used for simultaneously carrying out quantitative determinations on deoxyribonucleic acid (DNA) segments of cytomegalovirus (CMV) and BK virus (BKV). The kit comprises reagent A, B, C, D, E, and G. The reagent A is composed of a mixed set of primers and probes, wherein the primers and probes are targeted at consensus sequences ofCMV DNA and BKV DNA. The reagent B is a buffer solution containing dNTP. The reagent C is Tag DNA polymerase. The reagent D is composed of standard samples with different concentrations. The reagent E and G are respectively positive and negative controls. With the whole set of kit provided by the invention, quantitative determinations can be simultaneously carried out on DNA of CMV and BK virusesin clinical specimens of urine, blood or other body fluids.

Description

technical field [0001] The present invention relates to biotechnology, is a kind of molecular biology method based on quantitative nucleic acid amplification, particularly relates to using real-time fluorescent quantitative polymerase chain reaction (real-time PCR), by using specific primers to amplify cytomegalovirus (CMV ) and polyomavirus (BKV) conserved fragments, and quantify them by releasing fluorescent signals from specific probes. After optimizing the experimental conditions, a kit that can simultaneously detect CMV DNA and BKV DNA in a single reaction tube was constructed. Background technique [0002] CMV and BKV are latent viruses in kidney transplant recipients. When immunosuppressive therapy is overdone, both viruses reactivate to varying degrees and damage the transplanted kidney. These viruses can be found in the urine, plasma, and peripheral blood cells of kidney transplant recipients, and have a huge negative impact on the long-term survival of kidney tra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12N15/11
Inventor 陈必成白永恒杨亦荣
Owner THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL COLLEGE
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