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A kit and method for detecting human parvovirus b19

A human parvovirus and parvovirus technology, applied in the biological field, can solve the problem of high detection cost, and achieve the effects of good repeatability, good application prospects and high accuracy

Active Publication Date: 2016-01-13
CHENGDU RONGSHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Zhao Guoqiang et al., "Establishment of Fluorescent Quantitative PCR Detection Method for B19 Virus", Zhengzhou University Journal (Medical Edition), Volume 40, No. 3, May 2005, and other documents disclose methods for specific detection of B19 virus by QPCR. However, these methods are not Probes need to be used for detection to ensure the specificity and accuracy of the detection method, resulting in high detection costs

Method used

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  • A kit and method for detecting human parvovirus b19
  • A kit and method for detecting human parvovirus b19
  • A kit and method for detecting human parvovirus b19

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The design of embodiment 1 primer and the preparation of positive template

[0033] 1. Method

[0034] 1.1 Design and synthesis of primers

[0035] According to the sequence of the B19 virus on NCBI (NC_000883), primers were designed on the VP1 region of B19. The upstream primer (P1) was 5'-GGCACCTCTCAAAACACT-3' (SEQ ID NO: 1); the downstream primer (P2) was 5'-CCACTCCTTGCTGATACTC- 3' (SEQ ID NO: 2). Entrust Takara company to synthesize.

[0036] 1.2 Amplification of the B19 gene using the designed primers

[0037] 1.2.1 Nucleic acid extraction

[0038] The HighPureViralNucleicKit kit was used to extract B19-positive plasma and nucleic acid containing B19-positive plasma IVIG, and the nucleic acid of B19-negative plasma extracted by the kit was used as a negative control, and stored at -40°C.

[0039] 1.2.2 Amplification and identification

[0040] Using the genomic DNA of B19 as a template, using P1 and P2 as primers, PCR amplification:

[0041] Reaction system ...

Embodiment 2

[0054] The specific detection of embodiment 2 primers of the present invention

[0055] 1. Method

[0056] Extract the DNA of B19, PRV, BPV, MMV, and PK-15 as templates according to the method of 1.2.1 in Example 1, and perform PCR amplification. Three duplicate holes are set for each sample, and B19 (hole 1, hole 2, hole 3), PRV (hole 4, hole 5, hole 6), BPV (hole 7, hole 8, hole 9), MMV (hole 10, hole 11, hole 12), PK-15 (hole 13, hole 14, hole 15):

[0057] Reaction system (total volume 15 μl): EvaGreen master mix (SsoFast TM Supermix, 172-5201, Bio-rad company) 7.5 μl, upstream and downstream primers 0.5 μl, template DNA 1 μl (in ddH 2 O to make up 15 μl).

[0058] Reaction conditions: 95°C for 5min; 95°C for 10s, 52°C for 10s and 72°C for 10s, the last three steps were repeated for 40 cycles.

[0059] View the amplification curve.

[0060] 2. Results

[0061] like figure 2 As shown, fluorescence signals are collected at each cycle in the PCR process. As the num...

Embodiment 3

[0064] The sensitivity detection of embodiment 3 primers of the present invention

[0065] 1. Method

[0066] Dilute the B19 high-concentration positive plasma as shown in Table 1 to obtain samples containing different titers of B19;

[0067] According to the method of 1.2.1 in Example 1, sample nucleic acid was extracted respectively as a template, and PCR amplification was repeated 3 times at different times, and 8 parallel tubes were made at each concentration:

[0068] Reaction system (total volume: 15 μl): EvaGreen master mix 7.5 μl, upstream and downstream primers 0.5 μl, template DNA 1 μl (15 μl supplemented with ddH2O).

[0069] Reaction conditions: 95°C for 5min; 95°C for 10s, 52°C for 10s and 72°C for 10s, the last three steps were repeated for 40 cycles.

[0070] Check the amplification curve, if there is an amplification reaction curve within 40 CP values, it is counted as detected, and if there is no amplification reaction curve within 40 CP values, it is counte...

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Abstract

A kit for detecting human Parvoviridae B19, the kit comprising a primer pair shown by nucleotide sequence such as SEQ ID NO: 1-2, and amplifying the genes from human Parvoviridae B19. The kit can amplify gene fragments of human Parvoviridae B19 and has strong specificity, high sensitivity, good repeatability, and is used for quick testing of blood products and has good application prospects.

Description

technical field [0001] The invention relates to a kit for detecting human parvovirus B19, which belongs to the field of biotechnology. Background technique [0002] Human parvovirus B19 (Humanparvovirus B19) is a non-enveloped single-stranded DNA virus with a diameter of about 18-26nm. fetal death. Generally spread through the respiratory tract, but also through blood transfusion, blood products and blood-fetal route. Human parvovirus B19 has a small diameter and no envelope, so it is very difficult to inactivate and remove it. Therefore, it is necessary to establish a B19 detection method to effectively detect whether blood products contain B19, so as to improve the safety of blood products. [0003] At present, B19 detection methods are divided into three major technologies: serology, molecular biology and histology. Among them, serological tests represented by ELISA and molecular biological tests represented by PCR are relatively easy. However, because the B19 virus i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701
Inventor 郑朝共许锬容新宗黄琰
Owner CHENGDU RONGSHENG PHARMA
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