Treble real-time fluorescence quantitative polymerase chain reaction (PCR) method for simultaneously detecting epstein-barr virus (EBV), polyma virus (BKV) and cytomegalovirus (CMV) of people and kit

A real-time fluorescence quantitative, CMV-RP technology, applied in the field of medical molecular biological detection

Active Publication Date: 2012-02-29
同昕生物技术(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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  • Treble real-time fluorescence quantitative polymerase chain reaction (PCR) method for simultaneously detecting epstein-barr virus (EBV), polyma virus (BKV) and cytomegalovirus (CMV) of people and kit
  • Treble real-time fluorescence quantitative polymerase chain reaction (PCR) method for simultaneously detecting epstein-barr virus (EBV), polyma virus (BKV) and cytomegalovirus (CMV) of people and kit
  • Treble real-time fluorescence quantitative polymerase chain reaction (PCR) method for simultaneously detecting epstein-barr virus (EBV), polyma virus (BKV) and cytomegalovirus (CMV) of people and kit

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Experimental program
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Effect test

Embodiment 1

[0048] Design and system optimization of three kinds of viral primers and Taqman probes in embodiment 1

[0049] According to the genome sequences of CMV, EBV and BKV published by NCBI, using Primer Express 3 (Applied Biosystems) and Primer Premier 5 (Premier Biosoft International) primer design software, the designed primers and probe sequences are shown in Table 1:

[0050] Table 1 The designed detection primers and fluorescent labels

[0051]

[0052]

[0053] The fragments amplified by the three primers are shown in Seq ID No.1-3

[0054] HEX, BHQ1, ROX, BHQ2 and FAM marked at both ends of the Taqman probe in Table 1 are fluorescent markers and quenching groups of Taqman probes commonly used in this field, and their full names are as follows:

[0055]

[0056] The above primers and probes were synthesized by Shanghai Bioengineering Co., Ltd.

Embodiment 2

[0057] Embodiment 2. Cloning of three kinds of viral gene sequences

[0058] The basis of absolute quantification of quantitative PCR is to construct a standard curve. According to the sequence of the designed Taqman probe primer, design PCR primers at its two ends. Take ATCC (American Standard Biological Collection Center) CMV plasmid, BKV virus, and BKV virus standard plasmid as The template is amplified, and the amplified product is cloned into a T vector, transformed and identified. Finally, a plasmid standard cloned with the target sequence is obtained. Then it was quantitatively diluted to construct a standard curve. The specific operation steps are as follows: 1 Design of target sequence cloning primers

[0059] According to the genome sequences of the three viruses in Genebank, combined with the designed Taqman probe primer sequences, using the Primer Premier 6 software, design the cloning primers of the three viruses, and their sequences are shown in Table 2:

[00...

Embodiment 3

[0071] Example 3. Standard curve and multiple fluorescence real-time quantitative PCR (Triplex Real-time PCR) system establishment

[0072] Dilute the three constructed plasmid standards to 1,000,000 copies / ul, 100,000 copies / ul, 10,000 copies / ul, 1,000 copies / ul, and 100 copies / ul as amplification templates to establish multiple amplification systems and single amplification systems, respectively. Increase the system, the system ratio is as follows:

[0073] Triple system:

[0074]

[0075]

[0076] single system:

[0077]

[0078] The amplification reaction conditions are:

[0079]

[0080] Fluorescence quantitative PCR instrument: MJ Research Chromo 4, analysis software, Opticon Monitor Version 2.03, MJ Research

[0081] Results: as shown in Table 3 and figure 1 As shown, the Ct values ​​of the same concentration of the standard substance of each gene in the single amplification system and the multiple amplification system are basically the same, indicating ...

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Abstract

The invention discloses a treble real-time fluorescence quantitative polymerase chain reaction (PCR) method for simultaneously detecting an epstein-barr virus (EBV), a polyma virus (BKV) and a cytomegaloviruses (CMV) of people and a kit, which belong to medical molecular biological detection techniques. Three sets of primers and probes of the three viruses, which are designed by self, are adoptedto simultaneously detect a specimen to be detected. Experiments show that through adopting the three sets of probes and primers to simultaneously amplify a template to be detected or a manually-configured verification standard substance, the stocks of the three viruses in the specimen can be reflected peculiarly and correctly, and the three viruses cannot interfere with one another.

Description

technical field [0001] The invention relates to medical molecular biological detection technology. In particular, a triple real-time fluorescence quantitative PCR method and a kit for simultaneously detecting human EBV, BKV and CMV. It can be applied to the simultaneous monitoring of three viral loads in serum or other samples of post-transplant patients or patients in other immunosuppressed states. technical background [0002] Organ transplantation is one of the landmark disciplines of modern medicine. Since the first successful kidney transplant in 1959, more than 700,000 uremia patients in the world have received organ transplants and gained new life, and this number is still increasing at a rate of nearly 50,000 per year. As of 2003, my country has completed more than 50,000 cases of kidney transplantation, and now more than 5,000 cases are performed every year, ranking second in the world after the United States. There are more than 1 million uremia patients in Chin...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 李全焦守恕
Owner 同昕生物技术(北京)有限公司
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