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Nucleic acid sequence for isothermal amplification detection of measles virus, kit, detection method and application

A technology for constant temperature amplification detection and measles virus, which is applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc. It can solve the problems of false positives and high investment costs, and achieve simple steps and applicability Good, to ensure the effect of accuracy

Pending Publication Date: 2020-07-14
苏秀兰 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PCR technology has the advantages of high detection sensitivity, simple and fast operation, etc. However, its initial investment cost is relatively large (depending on the functional partition of the laboratory and the PCR instrument), and it is prone to false positives caused by laboratory contamination.

Method used

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  • Nucleic acid sequence for isothermal amplification detection of measles virus, kit, detection method and application
  • Nucleic acid sequence for isothermal amplification detection of measles virus, kit, detection method and application
  • Nucleic acid sequence for isothermal amplification detection of measles virus, kit, detection method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] The composition of the measles virus constant temperature amplification detection kit is as follows:

[0076] ①RNA extraction reagent: German Qiagen virus RNA extraction kit (Qia-52904);

[0077] ②Isothermal amplification reaction solution: 20mM Tris-HCl (pH=8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 1MMgSO 4 , 10×Thermo pol buffer, 5M betaine, 8U / μl Bst DNA polymerase, 10U AMV reverse transcriptase, 10mMdNTPs, 25×RNA secure, 0.1μM each of SEQ ID No.1 and SEQ ID No.2, SEQ ID No .3 0.3 μM, SEQ ID No.4 0.3 μM, SEQ ID No.5 0.6 μM; the primers and probes involved were all synthesized in Shanghai Sangon Bioengineering Co., Ltd., and the sequences are shown in Table 1.

[0078] ③ Positive control template: it is the in vitro transcription product containing the N gene fragment of measles virus. The preparation method of the positive control template is as follows: the genomic RNA of measles virus is amplified by RT-PCR with the downstream strand displacement primer and the ...

Embodiment 2

[0081] The specific method of the kit to detect measles virus:

[0082] The first step is to extract viral RNA from the specimen to be tested with RNA extraction reagent;

[0083] In the second step, add the RNA extracted in step 1 as a template into the PCR tube containing the constant temperature amplification reaction solution, and add the positive control template and negative control into the control PCR tube respectively, wherein both the sample RNA and the control RNA take 2 μl, The reaction solution was 18 μl, and the total reaction volume was 20 μl; the amplification reaction was carried out at 60°C for 90 minutes, and then the enzyme was inactivated at 80°C for 2 minutes;

[0084] The third step is to put the reacted PCR tube into the nucleic acid test strip anti-pollution detection device for detection, and interpret the experimental results after 5 minutes: when the quality control line (C line) and the detection line (T line) of the test strip are both positive w...

Embodiment 3

[0085] The optimization of embodiment 3 peripheral primers

[0086] Prepare kit according to the composition of embodiment 1, difference is:

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Abstract

The invention relates to the field of virus detection, in particular to a nucleic acid sequence for isothermal amplification detection of measles virus, a kit, a detection method and application. Thenucleic acid sequence comprises strand displacement primer sequences shown as SEQ ID No. 1 and SEQ ID No. 2, probes shown as SEQ ID No. 3 and SEQ ID No. 4, and a cross amplification primer sequence shown as SEQ ID No. 5. The kit and the detection method have the technical advantages of good specificity, high sensitivity, simple steps and high repeatability.

Description

technical field [0001] The invention relates to the field of virus detection, in particular to a nucleic acid sequence, kit, method and application for constant temperature amplification and detection of measles virus. Background technique [0002] Following the global eradication of polio, the World Health Organization (WHO) has made the elimination of measles the next goal. With the unremitting efforts of various countries, the number and incidence of measles worldwide have shown a downward trend in recent years, but they have not yet reached the level of elimination. Measles is an acute respiratory infectious disease caused by measles virus. Measles virus (MV) belongs to the genus Morbillivirus in the Paramyxoviridae family and is highly contagious, often accompanied by fever, rash, cough, and conjunctivitis. The incidence rate is high, and it can cause measles and serious complications after infecting the human body, including giant cell pneumonia and inclusion body en...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844
Inventor 苏秀兰温永俊韩汶延段蓉郑利英
Owner 苏秀兰
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