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Detection primer group, detection reagent and sequencing library for detecting organ transplantation postoperative infection

A technology for detecting primers and detection reagents, applied in the field of medical detection, can solve the problems of limited detection range of pathogens and low detection efficiency, and achieve the effects of improving survival rate and quality of life, reducing missed detection, and improving detection efficiency.

Pending Publication Date: 2019-09-27
上海奥根诊断技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects in the prior art that the detection range of pathogens infected after organ transplantation is limited and the detection efficiency is low.

Method used

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  • Detection primer group, detection reagent and sequencing library for detecting organ transplantation postoperative infection
  • Detection primer group, detection reagent and sequencing library for detecting organ transplantation postoperative infection
  • Detection primer group, detection reagent and sequencing library for detecting organ transplantation postoperative infection

Examples

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Embodiment 1

[0051] This embodiment provides a detection primer set for detecting infection after organ transplantation, including a multiplex PCR primer set for CMV virus, EBV virus, BK virus, HBV virus and B19 virus, and a universal primer set. Wherein, the multiplex PCR primer set includes the first primer set for detecting CMV virus, the second primer set for detecting EBV virus, the third primer set for detecting BK virus, and the fourth primer set for detecting HBV virus , and a fifth primer set for detecting B19 virus.

[0052] The upstream primer of the multiplex PCR primer set has a first tag sequence at the 5' end and an upstream recognition sequence at the 3' end, and the downstream primer of the multiplex PCR primer set has a second tag sequence at the 5' end and a downstream recognition sequence at the 3' end. The nucleotide sequence of the first tag sequence is shown in SEQ ID NO.1, the nucleotide sequence of the second tag sequence is shown in SEQ ID NO.2, and the upstream r...

Embodiment 2

[0060] This embodiment provides a detection reagent for infection after organ transplantation, comprising the following components:

[0061] (1) Primer mixture I, comprising the multiplex PCR primer set that embodiment 1 provides, the upstream primer of multiplex PCR primer set and downstream primer mix with the molar ratio of 1: 1, make the concentration of each upstream primer and each downstream primer be 5 μM, the concentration of primer mixture I is 5 μM;

[0062] (2) Primer mixture II, comprising the universal primer set provided in Example 1, the upstream universal primer and the downstream universal primer of the universal primer set are mixed in a molar ratio of 1:1, so that the concentrations of the upstream universal primer and the downstream universal primer are both 5 μM, the concentration of primer mixture II is 5 μM;

[0063] (3) KAPA Hifi Enzyme Mix, KAPA Hifi Enzyme Mix contains DNA polymerase 0.5U / 25μL, MgCl 2 2.5mM, dATP 0.3mM, dCTP 0.3mM, dGTP 0.3mM and ...

Embodiment 3

[0065] This embodiment provides a method for constructing a sequencing library for detecting infection after organ transplantation, specifically, including the following steps:

[0066] S1, extract the genomic DNA or cell-free DNA of the sample to be tested

[0067] Use QIAGEN's DNA extraction kit (Cat. No.: 51304 / 55114) to extract genomic DNA or free DNA from the samples to be tested. The samples to be tested are blood samples or urine samples from patients undergoing organ transplantation. Patients undergoing organ transplantation are CMV virus and EBV. Virus, BK virus, HBV virus and B19 virus are all positively infected patients. The purity and concentration of the extracted DNA were determined using a fluorescence quantification instrument Qubit3.0, and the concentration of the extracted DNA was adjusted to 1-5 ng / μl.

[0068] S2, using the genomic DNA or free DNA extracted in step S1 as a DNA amplification template, using the detection primer set (22 upstream primers + 2...

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Abstract

The invention discloses a detection primer group for detecting organ transplantation postoperative infection. The detection primer group comprises a multiplex-PCR (Polymerase Chain Reaction) primer group and a general primer group for CMV (Cytomegalovirus), EBV (Epstein-Barr virus), BK (BK virus), HBV (Hepatitis B Virus) and B19 (Human Parvovirus19). The detection primer group is capable of detecting five viruses in the same reaction system under the same amplification condition, virus detection coverage is wide, detection efficiency is high, and specificity is high. Meanwhile, the detection primer group is capable of enabling PCR amplification products to be directly applied to high-throughput sequencing, and detection throughput and detection sensitivity are improved. The invention further discloses a detection reagent comprising the detection primer group. The invention further discloses a sequencing library for detecting organ transplantation postoperative infection and a construction method of the sequencing library. According to the construction method of the sequencing library, construction steps of the conventional library are effectively simplified, and construction efficiency is improved.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a detection primer set, a detection reagent, a sequencing library, and a method for constructing a sequencing library for detecting infection after organ transplantation. Background technique [0002] Organ transplantation is currently the best treatment for many end-stage organ diseases. Survival of allograft recipients has improved dramatically over the past few decades, partly because of immunosuppressive agents that reduce the incidence of acute rejection, and partly because of infectious complications in recipients after transplantation can be effectively prevented and treated. In recent years, organ transplantation from donation after cardiac death (DCD) donors has developed rapidly in China. DCD donor organs have a long warm ischemia time, poor organ quality, and a high risk of delayed functional recovery after surgery. Stronger immune induction and immunosuppr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C40B40/06C40B50/06C12R1/93
CPCC12Q1/701C12Q1/705C12Q1/706C12Q1/686C40B40/06C40B50/06C12Q2600/16C12Q2537/143
Inventor 不公告发明人
Owner 上海奥根诊断技术有限公司
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