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A constant temperature amplification kit for detecting fever with thrombocytopenia syndrome virus and its application

A constant temperature amplification and platelet technology, applied in the biological field, can solve the problem of inability to monitor nucleic acid reverse transcription steps, and achieve the effects of reducing the chance of contamination of amplification products, good signal-to-noise ratio, and good specificity

Active Publication Date: 2018-05-25
JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

② A constant temperature amplification detection kit and detection method for New Bunia virus, application number: 201410795993.6, which discloses a kit for rapid detection of New Bunia virus, which cannot monitor the reverse transcription step of nucleic acid

Method used

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  • A constant temperature amplification kit for detecting fever with thrombocytopenia syndrome virus and its application
  • A constant temperature amplification kit for detecting fever with thrombocytopenia syndrome virus and its application
  • A constant temperature amplification kit for detecting fever with thrombocytopenia syndrome virus and its application

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Experimental program
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Effect test

Embodiment 1

[0034] The composition of the constant temperature amplification detection kit for fever with thrombocytopenia syndrome virus in this embodiment is as follows:

[0035] ①RNA extraction reagent: German Qiagen virus RNA extraction kit (Qia-52904);

[0036] ②Isothermal amplification reaction solution: 20mM Tris-HCl (pH=8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 6mMMgSO 4 , 0.1% TritonX-100%, 8U Bst DNA polymerase, 10U AMV reverse transcriptase, 1M betaine, 10U RNase Inhibitor, dNTPs each 0.8mM, upstream and downstream strand displacement primers each 0.1μM, upstream probe 0.3μM, downstream probe 0.5 μM, cross-amplification primer 0.5 μM, the primers and probes involved were synthesized in Shanghai Sangon Bioengineering Co., Ltd., the sequence is as follows:

[0037]Upstream strand displacement primer: 5′-TGGATGTTGGCCACTCTC-3′;

[0038] Downstream strand displacement primer: 5′-TTCTTCTGATTCAGAACA-3′;

[0039] FITC-labeled probe at the upstream 5′ end: 5′-FITC-GAGAGCTCATCCAAGGAT-3...

Embodiment 2

[0049] Sensitivity Test of Detection of Fever with Thrombocytopenia Syndrome Virus Using the Kit of the Invention

[0050] After 10-fold gradient dilution of the in vitro transcribed RNA standard product containing the M gene fragment of fever with thrombocytopenia syndrome virus, it is detected with the kit and detection method of the present invention, and the results are shown in figure 2 , where 1~8 are 10 respectively 7 ~10 0 Copy RNA standard product, 9 is a negative control, the result shows that the detection sensitivity of the kit of the present invention to fever with thrombocytopenia syndrome virus nucleic acid is 100 copies of viral RNA molecules / reaction, which can meet the requirements of rapid detection of heat with thrombocytopenia syndrome virus requirements.

Embodiment 3

[0052] Specific test for detection of fever with thrombocytopenia syndrome virus using the kit of the present invention

[0053] The viral nucleic acid related to fever with thrombocytopenia syndrome virus is detected genetically or clinically by using the kit and detection method of the present invention, and the results are as follows: image 3 As shown, 1 is a negative control, and 2 to 8 are Hantaan virus, Seoul virus, dengue virus, Anaplasma phagocytophilum, Leptospira, Xinjiang hemorrhagic fever virus, and fever with thrombocytopenia syndrome virus. The results show that, The detection kit of the invention has high specificity for detecting fever with thrombocytopenia syndrome virus.

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Abstract

The invention relates to a reagent kit and application thereof, particularly relates to an isothermal amplification reagent kit for detecting fever and thrombocytopenia syndrome viruses and application of the isothermal amplification reagent kit, and belongs to the field of biotechnologies. The isothermal amplification reagent kit for detecting the fever and thrombocytopenia syndrome viruses contains oligonucleotide and comprises RNA (ribonucleic acid) extraction reagents (1), isothermal amplification reaction liquid (2), positive control templates (3) and negative control components (4). The isothermal amplification reaction liquid (1) comprises 1*Bst Buffer, betaine, MgSO<4>, dNTPs, RNase inhibitors, Bst DNA (deoxyribonucleic acid) polymerase, AMV reverse transcriptase, sterile deionized water, upstream and downstream strand displacement primers, upstream and downstream probes and a cross amplification primer; the positive control templates (3) are in-vitro transcription products of M gene segments of the fever and thrombocytopenia syndrome viruses; the negative control components (4) are sterile deionized water. The isothermal amplification reagent kit and the application have the advantages that the isothermal amplification reagent kit is high in detection sensitivity, specificity and repeatability, simple in operation step and short in reaction time, and has a broad application prospect, integral reaction procedures can be carried out at the same temperatures, the fever and thrombocytopenia syndrome viruses can be detected only by the aid of a single isothermal instrument, and the like.

Description

technical field [0001] The invention relates to a kit and its application, in particular to a constant temperature amplification kit for detecting fever with thrombocytopenia syndrome virus and its application, belonging to the field of biotechnology. Background technique [0002] Fever with thrombocytopenia syndrome (Severe fever with thrombocytopenia syndrome, SFTS) is an important emerging infectious disease that threatens the health of our people and public health safety. The mortality rate is as high as 15-30%. SFTS is easily confused with blood, digestive, respiratory and other multi-system diseases, and clinicians lack awareness of it, which brings difficulties to the diagnosis and differential diagnosis of the disease. Fever with thrombocytopenia syndrome virus (SFTS virus, SFTSV) needs to be confirmed by laboratory means. Commonly used methods include virus isolation and culture, viral nucleic acid detection and immunological detection. Virus isolation and immunol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2565/625
Inventor 葛以跃崔仑标赵康辰祁贤史智扬周明浩胡林尤其敏
Owner JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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