Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method for three genotypes of human parvovirus B19, as well as universal detection primer, TaqMan probe and kit thereof

A real-time fluorescence quantitative and detection kit technology, applied in TaqMan probes and kits, real-time fluorescence quantitative PCR detection, and molecular biology detection of nucleic acids, to achieve the effects of programmed operation, convenient sampling, and simple and easy-to-use operation procedures.

Active Publication Date: 2013-12-18
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, commonly used detection techniques are only for type 1 of B19 virus, and cannot detect all genotypes of B19 virus

Method used

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  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method for three genotypes of human parvovirus B19, as well as universal detection primer, TaqMan probe and kit thereof
  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method for three genotypes of human parvovirus B19, as well as universal detection primer, TaqMan probe and kit thereof
  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method for three genotypes of human parvovirus B19, as well as universal detection primer, TaqMan probe and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. Design of real-time fluorescent quantitative PCR detection primers and TaqMan probes for simultaneous qualitative and quantitative detection of the three genotypes of human parvovirus B19 (B19 virus)

[0050] According to the gene sequence of the three genotype representative strains of B19 virus in GenBank (GenBank NO.: prototype: M13178; Lali: AY044266; V9: NC-004295), the DNA Man software is used for comparison, and the primers and TaqMan probes are designed In principle, the 121bp NS1 gene conservative region sequence (as shown in SEQ ID NO: 4 in the sequence list) is selected as the detection sequence, and real-time fluorescent quantitative PCR detection primers and TaqMan probes are designed to simultaneously detect the three genotypes of B19 virus to obtain the sequence as follows:

[0051] Upstream primer P (NS1-1): 5'-CGGGACCAGTTCAGGAGAAT-3' (SEQ ID NO: 1 in the sequence table), with a Tm value of 57.4°C;

[0052] Downstream primer P(NS1-2): 5'-CCCAACTAACA...

Embodiment 2

[0057] Example 2. Using the primers of the present invention to perform routine PCR detection on the three genotypes of human parvovirus B19

[0058] 1. Extract the DNA of B19 virus

[0059] The B19 virus international standard plasma (NIBSC code: 09 / 110) containing the three genotypes was used as the test sample (three samples), and the B19 virus was extracted with the High Pure Viral Nucleic Acid Kit (Roche) and referring to the kit instructions DNA.

[0060] 2. Routine PCR detection

[0061] Using the B19 viral nucleic acids of different genotypes extracted in step 1 as templates, PCR amplified 121bp of the conservative region of the NS1 gene under the guidance of the primers P(NS1-1) and P(NS1-2) of the present invention, and 25μL PCR reaction system It is: 10×PCR buffer (formulation: Tris·HCl (pH8.3) 100mM, KCl500mM, MgCl 2 15mM) 2.5μL, dNTPs (2.5mM each) 2μL, 400nM P(NS1-1), 400nM P(NS1-2), DNA template 5μL, 5U Taq enzyme (TaKaRa), add ddH 2 O to 25μL. The PCR reaction conditi...

Embodiment 3

[0062] Example 3. Real-time fluorescent quantitative PCR detection of B19 virus with primers and TaqMan probes of the present invention

[0063] 1. Extraction of B19 virus DNA

[0064] Take two raw plasma and one blood product as the sample to be tested, use the High Pure Viral Nucleic Acid Kit (Roche) and refer to the kit instructions to extract the sample DNA.

[0065] 2. Preparation of real-time fluorescent quantitative PCR standards

[0066] The target gene fragment for constructing the positive standard quality particle of the present invention is the 121bp B19 virus NS1 gene conserved region, the cloning vector is the pUC19 vector, and the vector size is 2686bp, Amp + For resistance, connect the B19 virus NS1 gene conserved region into the pUC19 vector multi-cloning site between SalI and BamHI restriction sites, and name the recombinant plasmid carrying the B19 virus NS1 gene conserved region B19-pUC19. This sequence was evaluated by Primer Premier5.0 software, and the full B19-...

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Abstract

The invention discloses a real-time fluorescent quantitative PCR (polymerase chain reaction) detection method which can be used for simultaneously performing qualitative and quantitative detection on three genotypes of human parvovirus B19, as well as a universal detection primer, a TaqMan probe and a kit thereof. The universal detection primer and the TaqMan probe disclosed by the invention can be combined with real-time fluorescent quantitative PCR to realize the purpose of accurately, qualitatively and quantitatively detecting B19 virus DNA (deoxyribonucleic acid) of the three genotypes of a sample to be detected, a formed NAT (nucleic acid testing) detection method against the three genotypes of B19 virus on the basis is obviously better than the existing detection technology, and the real-time fluorescent quantitative PCR detection method has the advantages of high accuracy, low pollution, fast detection speed, low requirements on instruments and equipment, low cost and the like; in addition to clinical detection and blood screening, the real-time fluorescent quantitative PCR detection method can also be used for performing qualitative and quantitative analysis on pollution status of the B19 virus in raw material plasma and blood products in scientific research and production and further has important significance in ensuring virus safety of the blood products.

Description

Technical field [0001] The present invention relates to a nucleic acid molecular biology detection method in the field of biotechnology, in particular to a real-time fluorescent quantitative PCR detection method for three genotypes of human parvovirus B19, and its universal detection primers, TaqMan probes and kits. Background technique [0002] Human parvovirus B19 (B19 virus) is closely related to the safety of blood transfusion and the virus safety of blood products. The B19 virus is currently the only parvovirus that can cause human diseases. The clinical manifestations of B19 virus infection are diverse. Infectious erythema (EI) is more common in children with B19 virus infection, also known as the "fifth disease." Adult infections are usually hidden infections, or only mild fever, headaches and other cold-like symptoms. Infections in patients with immune deficiencies can cause chronic anemia, infections in blood patients can cause transient aplastic crisis (TAC), and infe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 马玉媛章金刚贾俊婷赵雄郭逸吕茂民尹惠琼
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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