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Fluorescent quantitative PCR kit for detecting rubella viruses

A rubella virus and fluorescent quantitative technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as non-coverage, missed diagnosis, and false positive test results, so as to improve reliability and prevent false positives. Negative, easy-to-storage effect

Pending Publication Date: 2020-04-07
WUHAN BIOTECH GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The extraction of viral nucleic acid has a process of cracking, and pure nucleic acid like PCR products, cloned plasmids, and artificially synthesized nucleic acid fragments do not go through this process, so it cannot truly reflect the extraction process of viral nucleic acid, and it cannot Really realize the significance of quality control products
[0005] The shortcomings of the existing rubella virus detection technology using fluorescent quantitative PCR method mainly include the following points: (1) it does not cover all genotypes of common rubella virus in my country, so there is a situation of missed diagnosis; (2) in most cases, the sample response There is no internal control (internal standard) in the tube to control the false negative results that may be caused by inhibition of amplification in the tube. Even if an internal standard is set, it is generally plasmid DNA, which cannot truly and effectively reflect the extraction process of viral RNA; ( 3) Amplified products are easily contaminated by laboratory aerosols, and the test results may appear false positive

Method used

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  • Fluorescent quantitative PCR kit for detecting rubella viruses
  • Fluorescent quantitative PCR kit for detecting rubella viruses
  • Fluorescent quantitative PCR kit for detecting rubella viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] This embodiment provides a fluorescent quantitative PCR kit for detecting rubella virus, which includes a PCR reaction solution, an internal standard, a positive quality control product and a negative quality control product.

[0038] The PCR reaction solution includes upstream primer 1, downstream primer 1 and fluorescent probe 1 for detecting rubella virus, upstream primer 2, downstream primer 2 and fluorescent probe 2 for detecting internal standard, reverse transcriptase, RNase inhibitor, Taq DNA polymerase, UNG enzyme, dUTP and dNTPs.

[0039] The nucleotide sequence of the upstream primer 1 is 5'-TGCAGATGGCGCCCAGAGTGAG-3' (SEQ ID NO.1), and the nucleotide sequence of the downstream primer 1 is 5'-ACCGGCGTGGTGTATGGCACAC-3' (SEQ ID NO.2);

[0040] The base sequence of fluorescent probe 1 is 5'-TGGGCAGCAGCCCACTCCGCCC-3' (SEQ ID NO.3), its 5' end is marked with a fluorescent group FAM, and its 3' end is marked with a quenching group capable of quenching the fluorescen...

Embodiment 2

[0047] In this example, the kit described in Example 1 is used to detect the rubella virus RNA in the sample. The sample to be tested can be a sample in any form of whole blood, serum, plasma or amniotic fluid. The specific operation steps are as follows:

[0048] S1. Extract sample RNA

[0049] Aspirate 200 μL of the sample to be tested and add 5 μL of internal standard for nucleic acid extraction; take 25 μL of positive quality control and negative quality control, add 5 μL of internal standard respectively, and add physiological saline to 200 μL for nucleic acid extraction.

[0050] The virus nucleic acid extraction kit of Baitai Genomics (Ehan Jibei No. 20170246) was used to extract the nucleic acids in the samples to be tested (including internal standards), positive quality controls (including internal standards) and negative quality controls (including internal standards), For the specific operation method, please refer to the instruction manual of the kit.

[0051] S2...

Embodiment 3

[0067] Select an RV-positive serum sample with a clinically known concentration and divide it into 2 equal parts, one of which is added with RV-negative normal serum numbered as sample 1, and the other is added with RV-negative severe hemolysis (hemoglobin concentration greater than 5.0g / L ) serum number is sample 2, and the silica gel membrane method is used to extract its nucleic acid respectively as a template, and the primers and probes designed by the present invention are used to carry out real-time fluorescent PCR reaction, and the amplification results are shown in image 3 .

[0068] like image 3 As shown, the amplification Ct values ​​of sample 1 and its internal standard are within the specified range, and the amplification curve is good, but the amplification Ct value of sample 2 is obviously delayed compared with that of sample 1, and the amplification Ct value of sample 2 internal standard exceeds If the specified range is exceeded, the amplification curve does...

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Abstract

The invention discloses a fluorescent quantitative PCR kit for detecting rubella viruses. The kit comprises a PCR reaction solution, an internal standard, a positive quality control material and a negative quality control material, wherein the PCR reaction solution comprises an upstream primer, a downstream primer and a fluorescent probe which are used for detecting rubella virus RNA, and the internal standard material and the positive quality control material are respectively RNA pseudovirus containing chlamydia trachomatis tryptophan synthetase beta subunit encoding gene and RNA pseudoviruscontaining rubella virus specific conservative gene segment. The kit provided by the invention can detect all genotypes of rubella viruses published in rubella epidemiology in China, adopts pseudoviruses as internal standards, can truly and effectively carry out quality control on possible false negative detection results, and greatly improves the detection sensitivity, accuracy and stability.

Description

technical field [0001] The invention relates to the field of virus gene detection, in particular to a fluorescent quantitative PCR kit for detecting rubella virus. Background technique [0002] Rubella virus (Rubella virus, RV) is the only member of the Rubellavirus genus in the family Togaviridae, and has no cross-antigens with other viruses of the Togaviridae family. RV is a single-stranded positive-sense RNA virus that is transmitted through the respiratory tract. Humans are its only natural host and are distributed worldwide. Rubella itself is not serious, but the greatest threat to public health from RV is its teratogenicity. Pregnant women, especially pregnant women in the first trimester, infection of rubella virus can lead to fetal congenital rubella syndrome (Congenital rubella syndrome, CRS). Rubella virus can cause fetal infection through the placenta, and can reproduce in some tissue cells of the fetus, but does not kill the cells, eventually leading to abnorma...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2563/107C12Q2545/114
Inventor 熊慧杨雪婷肖樊许威李志杰瞿佳莉高峰英刘姣荣
Owner WUHAN BIOTECH GENE ENG
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