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Kit for LAMP detection of BK virus

A kit and virus technology, which is applied in the field of molecular biology and nucleic acid detection, can solve the problems of inability to conduct horizontal comparison, long detection cycle, and large difference in results, and achieve simple reaction result judgment method, easy operation, and high sensitivity Effect

Pending Publication Date: 2022-03-01
ZHONGSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of unified detection standards, different detection systems have caused large differences in results, and horizontal comparisons cannot be made. A consensus cannot be reached on the threshold value of virus copy number, which brings some troubles to the interpretation of the report.
Most of the existing methods for detecting BKV have a long detection cycle, slow speed, and low detection accuracy, so we urgently need products that can achieve fast, effective and accurate detection of BKV for the detection of BKV

Method used

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  • Kit for LAMP detection of BK virus
  • Kit for LAMP detection of BK virus
  • Kit for LAMP detection of BK virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 LAMP primer set design and optimization

[0044] 1.1 LAMP primer design

[0045] According to the VP1 gene sequence of the conserved region of BK virus reported on NCBI (accession number GenBank: AB365176.1, SEQ ID NO: 27), using the LAMP primer design software Primer Explorer V5 (http: / / primerexplorer.jp / lampv5e / index .html) designed 5 sets of specific primers BK1, BK2, BK3, BK4, BK5, primers and fluorescent markers

[0046] Record the probe sequence (5'-3') in the following table 1:

[0047] Table 1. BK virus detection primer set

[0048]

[0049] 1.2 Screening of LAMP primers

[0050] Through sensitivity comparison, the 5 primer sets designed in Example 1.1 were optimized. The specific method is as follows: according to the five sets of primers designed in Example 1.1, use five sets of primers to configure the isothermal amplification system respectively, and the The plasmid pcDNA3.1(+) of VP1 gene (GenBank: AB365176.1) (synthesized by Sangon Bioengi...

Embodiment 2

[0056] Example 2 Functional verification of BK2 primer set

[0057] Fluorescent quantitative PCR detection of BKV DNA in blood and urine is considered to be the most suitable non-invasive diagnostic method for monitoring the occurrence of BKVN in patients after renal transplantation. Therefore, according to the prior art document 1 report (Leung AY et al., Quantification of polyoma BK viruria in hemorrhagic cystitis complicating bone marrow transplantation, Blood, Volume 98, No. 6, 2001), the fluorescent quantitative PCR method is used for known positive urine The samples were further verified to illustrate the clinical detection effectiveness of the isothermal amplification method. The positive control is the target gene ribonucleic acid fragment containing BK virus, and the negative control is nuclease-free water.

[0058] 2.1 Extraction of DNA from the urine sample to be tested:

[0059] The specific operation of extracting the DNA of the urine sample to be tested by the ...

Embodiment 3

[0071] Example 3 Sensitivity test comparison of BK2 primer set and primer set reported in literature

[0072] After screening the primers, use the NanoDrop One / OneC Micro-Nucleic Acid Protein Concentration Analyzer to test the BKV genome standard substance (the plasmid pcDNA3.1(+) containing the target target VP1 gene (GenBank: AB365176.1), synthesized by Sangon Bioengineering Co., Ltd.) Measure the concentration, and then dilute into 10000copies / μL, 1000copies / μL, 100copies / μL, 10copies / μL, 1copies / μL samples in a 10-fold gradient, and use the samples of all dilution times as templates, respectively, using the LAMP detection reagent of this application Box (isothermal amplification method (experimental group) as described in Example 1.2) and prior art literature 2 report (Bipin Raj Bista et al., Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of BK Virus, JOURNAL OF CLINICAL MICROBIOLOGY , Vol. 45, No. 5, pp. 1581-1587) for the isothermal amp...

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Abstract

The invention provides a method for detecting a BK virus based on a loop-mediated isothermal amplification (LAMP) technology, a primer group for the method and a corresponding kit. The BK virus can be subjected to specific loop-mediated isothermal amplification by utilizing the primer group. The method disclosed by the invention is good in specificity, high in sensitivity, good in repeatability, simple to operate, convenient and rapid, can be well applied to identification of the BK virus, and is suitable for clinical and laboratory detection.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and nucleic acid detection, and in particular relates to a primer set, a kit and a detection method for isothermally amplifying BK virus based on a loop. Background technique [0002] Human BK virus (BKV) is a non-enveloped circular double-stranded DNA virus with a genome of about 5300 bp. It belongs to the polyomavirus family (polyomaviruses) with human JC virus (JCV) and monkey SV40 virus. The prevalence of BKV infection in the population exceeds 85%, but clinical symptoms are often limited to immunocompromised individuals. The primary infection of BKV mostly occurs in childhood, followed by latent infection in the urinary system, and latent infection of BKV is also found in lymphoid tissue, liver, lung, eye, brain and other places. BKV is an opportunistic pathogen. People with normal immunity have no symptoms of infection. When the immune function of the host is reduced or suppresse...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2563/107C12Q2537/1376C12Q2545/113
Inventor 朱同玉王吉妍
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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