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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 simple composition, multiple experimental information, and reduction of manpower and material resources

Inactive Publication Date: 2012-02-08
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

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A set of primers and probes for the quantitative detection of CMV and BKV DNA. Including a pair of primers against CMV comprising upstream primers SEQ ID NO.1 and downstream primers SEQ ID NO.2 and a probe SEQ ID NO.3; and a pair of primers against BKV comprising upstream primers SEQ ID NO.4 and downstream Primer SEQ ID NO.5, and a probe SEQ ID NO.6. SEQ ID NO.3 and SEQ ID NO.6 can be labeled with different fluorescent dyes for quantitative detection.

Embodiment 2

[0032] A standard product matching the method for detecting CMV and BKV DNA, simultaneously containing the purified plasmid of CMV and BKV two kinds of detection target DNA fragments SEQ ID NO.9. The preparation process obtains the target fragment after amplifying the CMV and BKV DNA and connects it to the T vector. Use the primers of SEQ ID NO.10 and SEQ ID NO.11 to amplify the CMV DNA. The program is 95°C for 2min; 94°C for 20sec, 60°C for 30sec, and 72°C for 30sec for a total of 35 cycles. The conditions are 200nM primers and Taq enzyme 50U / mL, 100μM dNTPs, 20mM Tris-HCl, 20mM KCl, 10mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 . The CMV target DNA fragment was obtained by the above method. PCR amplification of BKV DNA was performed using primers of SEQ ID NO.12 and SEQ ID NO.13. The program was 95°C for 2min; 94°C for 20sec, 60°C for 30sec, and 72°C for 30sec for a total of 35 cycles, and the condition was 200nM Primers, Taq enzyme 50U / mL, 100μM dNTPs, 20mM Tris-HCl, 20mM KCl, 10m...

Embodiment 3

[0034] A kit for quantitatively detecting the DNA of CMV and BKV, consisting of the following reagents:

[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).

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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 Applications(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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