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Detection method and kit of BK virus, and application thereof

A detection method and technology of a kit, applied in the field of BK virus detection, can solve problems such as inability to achieve, and achieve the effects of preventing contamination, good specificity and high sensitivity

Inactive Publication Date: 2016-11-02
BEIJING SEARCH BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

This patented technology describes methods and tools used during diagnostic testing involving detecting bacterial or fungi called Bacillus Calibaceria (BC). These techniques involve collecting blood from humans and analyzing it under special conditions like temperature changes. By measuring tiny amounts of these materials, researchers aimed towards understanding how they work better than existing tests. They found that certain compounds were more harmful when compared against others due to their ability to bind specifically to different parts of cells within our body. However, there was also evidence showing that some compound caused damage even if we didn't use them beforehand. Therefore, the technical effects of this new technique include improved accuracy, quicker analysis times, lower costs, and greater flexibility in disease management decisions based upon results.

Problems solved by technology

The technical problem addressed by this patented research relating to bovinemia caused by chronic viruses such as EBOV and CMV is their ability to induce severe complications like acute lung injury due to cytokogenesis induced by these diseases. Current treatments involve symptoms remission but they only work on some cases while others require hospitalization. Therefore there is a demand for accurate testing techniques capable of detecting disease severities early enough during medical interventions before surgery.

Method used

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  • Detection method and kit of BK virus, and application thereof
  • Detection method and kit of BK virus, and application thereof
  • Detection method and kit of BK virus, and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0054] This embodiment provides the method for obtaining the sample to be tested:

[0055] BKV DNA was extracted from human urine samples by magnetic bead method to obtain samples to be tested. The magnetic bead method extracts nucleic acid using a unique lysate and proteinase K to quickly lyse the cells and inactivate intracellular nucleases, then the genomic DNA is selectively adsorbed to the magnetic beads, and then through a series of rapid rinsing-separation steps, the inhibitor removal solution and washing solution to remove impurities such as cell metabolites and proteins, and finally the pure genomic DNA can be eluted from the magnetic beads with double distilled water.

[0056] 1. Reagents and components for extracting BKV DNA from human urine samples by magnetic bead method

[0057] (1) Lysis solution: 100mL dosage as an example, 40.47g of guanidine hydrochloride, 0.5132g of tris(hydroxymethyl)aminomethane, 0.0932g of sodium chloride, 6.78mL of Tween-20, add 16mL of...

Embodiment 2

[0077] Qualitative detection of BK virus

[0078] Present embodiment 2 provides a kind of qualitative detection method of BK virus:

[0079] 1. PCR amplification reaction of BKV DNA

[0080] 1) Reagents and components used in the PCR amplification reaction of BKV DNA

[0081] (1) BKV primer probe mixture:

[0082] The specific composition of the BKV primer-probe mixture is: upstream primer BKV-F (20 μM) 1 μL, downstream primer BKV-R (20 μM) 1 μL, Taqman fluorescent probe BKV-FP (10 μM) 0.8 μL, purified water 2.2 μL.

[0083] The preferred components of the BKV primer-probe mixture are: upstream primer BKV-F (20 μM) 1 μL, downstream primer BKV-R (20 μM) 1 μL, Taqman fluorescent probe BKV-FP (10 μM) 0.8 μL, internal standard upstream primer ( 20 μM) 0.5 μL, internal standard downstream primer (20 μM) 0.5 μL, internal standard probe (10 μM) 0.375 μL, purified water 0.825 μL.

[0084] The upstream primer BKV-F comprises the base sequence shown in SEQ ID NO:1;

[0085] The dow...

Embodiment 3

[0108] Present embodiment 2 provides a kind of quantitative detection method of BK virus:

[0109] 1. PCR amplification reaction of BKV DNA

[0110] 1) Reagents and components used in the PCR amplification reaction of BKV DNA

[0111] (1) BKV primer probe mixture:

[0112] The components of the BKV primer-probe mixture are: upstream primer BKV-F (20 μM) 1 μL, downstream primer BKV-R (20 μM) 1 μL, Taqman fluorescent probe BKV-FP (10 μM) 0.8 μL, internal standard upstream primer (20 μM ) 0.5 μL, internal standard downstream primer (20 μM) 0.5 μL, internal standard probe (10 μM) 0.375 μL, purified water 0.825 μL.

[0113] The said upstream primer BKV-F comprises the base sequence shown in SEQ ID NO:1;

[0114] The downstream primer BKV-R comprises the base sequence shown in SEQ ID NO:2;

[0115] The Taqman fluorescent probe BKV-FP comprises the base sequence shown in SEQ ID NO: 3, the fluorescent reporter group is 6-FAM; the fluorescent quencher group is BHQ1.

[0116] (2) P...

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Abstract

The application discloses a detection method of BK virus, which includes the steps of: extracting BKV DNA from human urine samples through a magnetic bead method to obtain a to-be-tested sample; performing a PCR amplification reaction; detecting a reaction result with a fluorescent quantitative PCR instrument, wherein during the collection of fluorescent signals, the fluorescent signals are collected at 60 DEG C by means of fluorescein which is corresponding to a 5'-terminal fluorescent group of a Taqman fluorescent probe BKV-FP. The invention also discloses a kit for detecting the BK virus. The detection method and the kit have high sensitivity and can reach the lowest detection limit of 500 copies/ml. The detection method has good specificity, has no cross reactions with other viruses, such as JC virus (JCV), hepatitis C virus (HCV), human cytomegalovirus (HCMV) and hepatitis B virus (HBV).

Description

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Claims

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Application Information

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Owner BEIJING SEARCH BIOTECH
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