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Primer probe combination and kit for detecting five important arthropod/ odonto-mediated viruses and application of primer probe combination and kit

A primer probe and virus detection technology, which is applied in the direction of recombinant DNA technology, microorganism-based methods, microorganism measurement/inspection, etc., can solve the problems of high reagent price, long detection time, and inability to achieve instant detection, etc., and achieve good application Effects of foreground, high sensitivity, and specificity

Pending Publication Date: 2020-10-27
SHENZHEN PEOPLES HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional fluorescent quantitative PCR method still has high reagent prices, long detection time, and cannot achieve real point-of-care testing (POCT), and cannot meet the requirements of portable, mobile, and on-site rapid inspection.

Method used

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  • Primer probe combination and kit for detecting five important arthropod/ odonto-mediated viruses and application of primer probe combination and kit
  • Primer probe combination and kit for detecting five important arthropod/ odonto-mediated viruses and application of primer probe combination and kit
  • Primer probe combination and kit for detecting five important arthropod/ odonto-mediated viruses and application of primer probe combination and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1 Repeatability Detection

[0090] The positive control of each virus diluted 10 times was used as the sample to be tested for repeated detection, and the detection was repeated 3 times.

[0091]1. Nucleic acid extraction (used with Hs480 heating centrifuge)

[0092] 1) Take 50 μl of the sample and add 5 μl FastLyse L4, shake and mix;

[0093] 2) Place the mixed sample in the HS480 heating centrifuge machine, heat at 95°C for 2min, and centrifuge at 5000rpm for 2min;

[0094] 3) After centrifugation, take the supernatant as the sample RNA, numbered;

[0095] 4) The positive control, negative control and samples to be tested are processed synchronously.

[0096] 2. Preparation of PCR reagents

[0097] 1) Prepare the PCR reaction solution: pre-mix the ultra-fast PCR buffer1 (68 μl) and the ultra-fast PCR enzyme system (17 μl) (2 more than the actual number of reaction tubes, to avoid loss during sample addition, and to avoid air bubbles during mixing);

[009...

Embodiment 2

[0115] Embodiment 2 specific detection

[0116] Each virus positive control was used as the sample to be tested for specific detection, and the detection method was the same as in Example 1.

[0117] Result: Each virus positive control sample was only positive under the corresponding probe, and the rest were negative, with good specificity. The results were as follows: Figure 1 to Figure 5 shown.

Embodiment 3

[0118] Example 3 Sensitivity Detection

[0119] Each virus positive control in the kit (concentration is 1 × 10 5 copies / ml) was serially diluted to 1×10 4 copies / ml, 1×10 3 copies / ml, 1×10 2 copies / ml and 1×10 1 Copies / ml, after preparation, use it as the sample to be tested for sensitivity testing with the corresponding kit.

[0120] Concrete implementation steps are with embodiment 1.

[0121] Results: The detection limits of YFV, LASV, HTV, CHIKV and DENV were not higher than 1×10 1 copies / ml, the result is as Figure 6 shown.

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Abstract

The invention relates to the field of biological detection, and particularly relates to a primer probe combination and kit for establishing a top-speed detection method for simultaneously detecting Yellow Fever Viruses (YFV), Lasa viruses (LASV), Hantaan viruses (HTV), chikungunya viruses (CHIKV) and dengue viruses (DENV) and application of the primer probe combination and kit. The primer probe combination and kit have high sensitivity and specificity, compared with other fluorescent quantitative PCR, the established method can obtain a result within half an hour, clinical rapid detection canbe achieved, real POCT is expected to be achieved, and the primer probe combination and kit have good application prospects.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a combination of primers and probes, a kit and an application thereof for the detection of five important arthropod / rodent vector-borne viruses. Background technique [0002] According to my country's experience in the prevention and control of emerging infectious diseases in recent years, especially severe acute respiratory syndrome (severe acute respiratory syndrome, SARS), influenza A (H1N1), imported Ebola, MERS in the Middle East, Zika, etc., it is shown that, Rapid diagnosis is playing an increasingly important role in the prevention and control of viral infectious diseases. In terms of methodology, these diagnostic reagents are mainly divided into two categories, namely, immunological diagnosis and molecular diagnosis. Immunological diagnostic techniques are generally detection techniques based on antigen-antibody specific reactions, mainly including enzyme-linked immun...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2563/107C12Q2563/159
Inventor 吴诗品金宁一李体远李昌杜寿文田明尧任琴张星艳王宇航戴少波
Owner SHENZHEN PEOPLES HOSPITAL
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