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PCR (Polymerase Chain Reaction) primer group for detecting human herpes virus 6B type in human body fluid and droplet type digital PCR kit thereof

A technology for detecting human and herpes virus, applied in microorganisms, recombinant DNA technology, microorganism-based methods, etc., can solve the problems of increasing the difficulty and necessity of differential diagnosis, increasing the risk of HHV-6B infection and viral encephalitis, etc. To achieve the effect of shortening the treatment cycle, low cost and less time-consuming

Pending Publication Date: 2022-07-29
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After CAR-T cell therapy, anti-interleukin-6 or interleukin-6 receptor monoclonal antibodies and high-dose corticosteroids are often used to treat CRS, which increases HHV-6B infection and viral infection in patients receiving CAR-T therapy. The risk of encephalitis, which increases the difficulty and necessity of differential diagnosis

Method used

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  • PCR (Polymerase Chain Reaction) primer group for detecting human herpes virus 6B type in human body fluid and droplet type digital PCR kit thereof
  • PCR (Polymerase Chain Reaction) primer group for detecting human herpes virus 6B type in human body fluid and droplet type digital PCR kit thereof
  • PCR (Polymerase Chain Reaction) primer group for detecting human herpes virus 6B type in human body fluid and droplet type digital PCR kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1. Extraction of cell-free DNA from body fluids

[0045] 1. Specimens such as peripheral blood, cerebrospinal fluid, and urine were filtered with a 300-mesh filter membrane and transferred to a 15ml centrifuge tube, centrifuged at 3000rpm for 5min in a low-speed centrifuge, and the supernatant was transferred to a new 15ml centrifuge tube, and centrifuged at 3000rpm for 10min to remove cells as much as possible. and other tangible substances.

[0046] 2. Extraction method of cell-free DNA: use the kit from QIAGEN from Germany to carry out cell-free DNA ( Circulating Nucleic Acid Kit) extraction, the specific operation is as follows:

[0047] 1) Add 5ml of centrifuged supernatant (take 5ml as an example) to a 50ml centrifuge tube, add 4ml (1:0.8) ACL lysis solution, 500μL proteinase K (1:0.1), 5μL Carrier RNA (0.2μg / μL) to it ), cover and shake for 30s;

[0048] 2) Put the 50ml centrifuge tube on the centrifuge tube rack, put it into a 60°C water bath and incubate fo...

Embodiment 2

[0057] 1. Extraction of genomic DNA (gDNA) from peripheral blood cells

[0058] 1.3-5ml of EDTA anticoagulated peripheral blood was lysed by erythrocyte lysate for 10min, centrifuged at 1500rpm for 5min, the supernatant was discarded, and the remaining cells were resuspended in 200μL PBS and transferred to a 1.5ml EP tube.

[0059] 2. Extraction method of cell gDNA: gDNA ( DNA BloodMini Kit) extraction, the specific operation is as follows:

[0060] 1) Add 20 μL of proteinase K and 200 μL of AL to a 1.5 ml tube, vortex and mix for 15 s, and centrifuge briefly with a small shaker to concentrate the liquid at the bottom of the tube;

[0061] 2) Incubate in a 60°C metal bath for 10min;

[0062] 3) Take out the EP tube from the metal bath, add 200 μL of absolute ethanol to it, shake and mix for 15s, and centrifuge briefly with a small shaker;

[0063] 4) Prepare the spin column and mark it, add the liquid in step 3) to it, 12000rpm for 1min, and replace with a new receiving tu...

Embodiment 3

[0068] Example 3 Design and screening of primer probes

[0069] 1. Design of primers and probes: The genome sequence of human herpesvirus 6 strain NY-380 was obtained based on the NCBI (National Center for Biotechnology Information) online tool, combined with the results of metagenomic sequencing of HHV-6B infected patients in our center , using Primer Express 3.0.1 software to design specific primers and probes suitable for digital PCR, the alternative primers and probes are as follows:

[0070] F1: 5'-TCCATTGTTTGATTGATTTCCGTAT-3'

[0071] R1: 5'-AACGCGGGATGTTCTATTGG-3'

[0072] Probe1: 5'-CTTGAGCTTGTAGATAAT-3'

[0073] F2: 5'-GTCAAAGCCGAGCTCTACAAAAC-3'

[0074] R2: 5'-GTGTCTGAGATGATGATGGGACCATT-3'

[0075] Probe2: 5'-ACAAAGAAGACGTGCTCC-3'

[0076] F3: 5'-AAGTTCCATTGTTTGATTGATTTCC-3'

[0077] R3: 5'-TAACGCGGGATGTTCTATTGG-3'

[0078] Probe3: 5'-TCTTGAGCTTGTAGATAAT-3'

[0079] 2. Screening of probes and primers

[0080] 1) Primer probe combination: F1R1P1, F2R2P2, F3R3...

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) primer group for detecting human herpes virus type 6B in human body fluid and a droplet type digital PCR kit thereof. A specific primer and an MGB hydrolysis probe are designed on the basis of a conserved sequence of a human herpes virus 6B genome, so that the microdroplet type digital PCR kit is obtained, free DNA in body fluid of a patient suffering from blood system diseases after being treated by chimeric antigen receptor T cells is extracted, HHV-6B DNA is detected through the microdroplet type digital PCR kit, and the detection sensitivity is high. The method has important guiding significance for assisting clinical understanding of HHV-6B infection conditions, identification of infection of other pathogens and identification of CAR-T cell-related encephalopathy syndromes.

Description

technical field [0001] The invention relates to the field of viral nucleic acid detection, in particular to a PCR primer set for detecting human herpesvirus 6B in human body fluid and a droplet type digital PCR kit. Background technique [0002] Human herpesvirus 6 (human herpesvirus 6, HHV-6) belongs to the β subfamily of the family Herpesviridae. It is a T-lymphocyte-tropic double-stranded DNA virus. After primary infection of HHV-6, it can be latent in the body for life, and 90% of adult Humans have latent HHV-6 infection and reactivation occurs in immunocompromised populations such as hematopoietic stem cell transplantation, organ transplantation, and AIDS patients. The proportion of reactivation in hematopoietic stem cell transplantation population reaches 30-50%. HHV-6 can be divided into two subtypes, HHV-6A and HHV-6B. HHV-6B is the most detected subtype in various body fluids of the human body and can cause various diseases. The infection rate is low, the primary i...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/705C12Q1/6851C12Q2600/166C12Q2531/113C12Q2563/159C12Q2545/101C12Q2563/107
Inventor 陈丽婷顾佳纪红艳周剑峰肖敏黄亮
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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