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Primer group and probe group capable of simultaneously detecting BK virus and JC virus, and purpose

A JC virus and primer set technology, applied in the field of medical molecular biological detection, can solve the problems of increasing the detection burden of patients, less diagnostic reagents, and high price, and achieve the effects of reducing detection costs, high specificity, and reducing reaction costs.

Pending Publication Date: 2021-03-19
苏州奥根诊断科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few diagnostic reagents for simultaneous detection of BKV and JCV on the market (Norgen, Inc.), and they are expensive, which increases the detection burden of patients, and separate detection is time-consuming and labor-intensive

Method used

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  • Primer group and probe group capable of simultaneously detecting BK virus and JC virus, and purpose
  • Primer group and probe group capable of simultaneously detecting BK virus and JC virus, and purpose
  • Primer group and probe group capable of simultaneously detecting BK virus and JC virus, and purpose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 A primer set and probe set for simultaneous detection of BK virus and JC virus

[0060] This embodiment screens out target sequences (as shown in SEQ ID NO.1 (BKV) and SEQ ID NO.2 (JCV)) from the conserved regions on the BKV and JCV genomes, and designs highly specific primer sets based on the above target sequences and probe sets, as follows:

[0061] The primer set includes upstream primer BJ-F and downstream primer BJ-R, the nucleotide sequence of upstream primer BJ-F is shown in SEQ ID NO.3, and the nucleotide sequence of downstream primer BJ-R is shown in SEQ ID NO.4 Show;

[0062] The probe set includes probe BK-P and probe JC-P, the nucleotide sequence of probe BK-P is as shown in SEQ ID NO.5, and the nucleotide sequence of probe JC-P is as shown in SEQ ID Shown in NO.6.

[0063] The 5' end of each probe in the above probe group is modified with a fluorescent reporter group, and the 3' end is modified with a fluorescent quenching group; the fluoresce...

Embodiment 2

[0069] Embodiment 2 A kind of fluorescent quantitative PCR kit that simultaneously detects BK virus and JC virus

[0070] This embodiment provides a fluorescent quantitative PCR kit for simultaneous detection of BK virus and JC virus, including the primer set and probe set in Example 1.

[0071] Further, the molar ratio of the upstream primer BJ-F, the downstream primer BJ-R, the probe BK-P and the probe JC-P is 1:1:0.5:0.5.

[0072] Further, it also includes at least one of PCR master mix, negative quality control, BKV DNA standard and JCV DNA standard;

[0073] The negative quality control substance is sterile water;

[0074] The BKV standard product is a recombinant plasmid cloned with a BKV target fragment (the nucleotide sequence shown in SEQ ID NO.1), and there are 6 concentration gradients, and the concentrations are 5×10 2 , 5×10 3 , 5×10 4 , 5×10 5 , 5×10 6 , 5×10 7 copies / uL;

[0075] The JCV standard product is a recombinant plasmid cloned with a JCV target ...

Embodiment 3B

[0085] Embodiment 3BKV and JCV fluorescence quantitative PCR standard curve formulation

[0086] Utilize the kit in embodiment 2 to prepare the system of the PCR amplification reaction of different standard items as:

[0087] PCR master mix, 12.5uL;

[0088] Upstream primer BJ-F, 10uM, 1uL;

[0089] Downstream primer BJ-R, 10uM, 1uL;

[0090] Probe BK-P, 10uM, 0.5uL;

[0091] Probe JC-P, 10uM, 0.5uL;

[0092] DNA template (standards of each dilution concentration of BKV or JCV, also includes 5×10 1 Copies / uL concentration of BKV or JCV standard, or negative quality control) 1uL;

[0093] Make up to 25uL with sterile water.

[0094] The conditions of the PCR amplification reaction were: pre-denaturation at 95°C for 5min; denaturation at 95°C for 10s, annealing and extension at 60°C for 40s, 40 cycles.

[0095] Fluorescent signals were collected at 60°C in each cycle;

[0096] The results of the fluorescent quantitative PCR amplification curve of BKV / JCV standard substan...

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Abstract

The invention relates to the field of medical molecular biology detection, in particular to a primer and a probe capable of simultaneously detecting a BK virus and a JC virus, and a purpose. The invention provides a primer group capable of simultaneously detecting the BK virus and the JC virus. The primer group is designed on the basis of nucleotide sequences disclosed by SEQ ID NO.1 and SEQ ID NO.2, the above target sequence can meet a primer design requirement in a range of 100-200bp, meanwhile, the primer designed on the basis of the target sequence can effectively authenticate the BK virusand also can accurately authenticate the JC virus, and difficulty to design a fragment suitable for the primer is high.

Description

technical field [0001] The invention relates to the field of medical molecular biological detection, in particular to a primer set and a probe set for simultaneously detecting BK virus and JC virus and their application. Background technique [0002] Polyomaviruses (PV) belong to the papapovirus family, mainly including BK virus (BKV), JC virus (JCV) and simian vacuolar virus 40 (SV40). Generally, there are no obvious clinical symptoms, and it can be latent in renal tubules and urothelial cells. It is not pathogenic to healthy people, but when the body's immunity is low, the virus in the latent body is activated, causing a series of clinical symptoms, such as progressive multifocal leukoencephalopathy (PML), polyomavirus-associated nephropathy ( PVAN) and so on. [0003] Kidney transplantation is an important means of treating end-stage renal disease. However, patients need to take immunosuppressants for a long time after transplantation to avoid the occurrence of rejecti...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 不公告发明人
Owner 苏州奥根诊断科技有限公司
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