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Novel coronavirus RPA test strip detection kit

A technology for detection kits and kits, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of no design standards, low success rate, and difficulty in manual design, etc., to reduce reagents Cost, detection time saving, high sensitivity effect

Pending Publication Date: 2020-09-08
CHENGDU HAIZHIYUAN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there is no RPA detection kit for the detection of new coronaviruses. The main reason is that the design of RPA primers is different from ordinary PCR or qRT-PCR. At present, there is no unified design standard, and it cannot be directly obtained by primer design software. more difficult and less successful

Method used

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  • Novel coronavirus RPA test strip detection kit
  • Novel coronavirus RPA test strip detection kit
  • Novel coronavirus RPA test strip detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 RPA kit (test strip method) composition and method of use

[0035] The main components of the kit are shown in Table 1.

[0036] Table 1 The main components of the kit

[0037]

[0038]

[0039] Note: Packaging specification: 48 times / box bag, you need to prepare your own virus nucleic acid extraction kit, RNase inhibitor (recommended: Takara: article number: 9766, 2313Q); nucleic acid detection test strip: composed of sample pad, cellulose film, Consisting of detection belt, control belt and absorbent pad, it is recommended to use Universal nucleic acid detection test strip or disposable nucleic acid detection device (model: D001-3) of Hangzhou Ustar Biotechnology Co., Ltd.

[0040] The detailed introduction of primers, probes and other reagents in Table 1 is as follows:

[0041] (1) Primers, probes

[0042] The primer and probe sequences of the present invention are shown in Table 1. In the probe sequence, the 5' is modified by the fluorescent ...

experiment example 1

[0059] Experimental example 1 Specificity and sensitivity detection of RPA kit for detection of novel coronavirus

[0060] 1. Sensitivity detection:

[0061] The concentration of the plasmid standard was 40ng / μL measured by a UV spectrophotometer, the base number of pUC57-KANA was 2710bp, the target fragment size was 253bp, and the average relative molecular mass of each base was 660 Daltons / bp. The copy number in is calculated according to the formula: sample copy number (copies / ul) = (6.02×10 23 )×plasmid concentration (ng / μL)×10 -9 / (660×base number), the final calculated copy number is 1.23×10 10 copies / ul. The above standard is diluted according to 10-fold ratio, and the concentration is taken at 10 7 -10 1 Copies / ul was used for the experiment, reacted at 39°C for 10 minutes, after the reaction was completed, 5 μL of the reaction liquid was drawn, diluted 10-50 times by adding 1×PBST, and put into a test strip for 5-20 minutes.

[0062] The result is as figure 2...

experiment example 2

[0066] Experimental Example 2 Primer Design

[0067] At present, there is no definite RPA primer sequence design rule, and no design software is available. The inventor's primer design mainly follows the following rules:

[0068] 1) Primers should be 30-35 bases in length for optimal formation of recombinase / primer filaments; longer primers (>45 bases) are not recommended;

[0069] 2) Long tracks of a specific nucleotide or a large number of small repeats (such as AAAAA) should be avoided;

[0070] 3) RPA can amplify long sequences up to 1.5kb, but better results can be obtained with shorter amplicons in the range of 80–400bp (optimally 100–200bp);

[0071] 4) Try to avoid primer-dimer and hairpin structures;

[0072] 5) Too high (>70%) or too low (<30%) GC content is not conducive to RPA amplification;

[0073] 6) For the three nucleotides at the 3' end, guanine and cytosine contribute to the stable combination of the polymerase, which can improve the amplification perform...

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Abstract

The invention discloses a novel coronavirus RPA test strip detection kit, and belongs to the field of virus detection. The kit disclosed by the invention comprises primers with sequences shown as SEQID NO.1 and SEQ ID NO.2, and a probe with a sequence shown as SEQ ID NO.3. The kit provided by the invention does not need to depend on a high-cost (fluorescent quantitative) PCR instrument, can realize high-sensitivity and specific rapid detection at reagent cost of 50% lower than that of a conventional RPA detection kit, and is suitable for novel coronavirus detection in various occasions.

Description

technical field [0001] The invention belongs to the field of virus detection. Background technique [0002] The new type of coronavirus, that is, SARS-Cov-2, the common signs of people infected with the coronavirus include respiratory symptoms, fever, cough, shortness of breath and dyspnea. The disease caused by the novel coronavirus is also known as "COVID-19," and in more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death. [0003] The detection of 2019-nCoV is an important part of epidemic prevention and control. Various detection kits are currently available, which can be divided into nucleic acid detection kits and protein detection kits according to the detection objects. Among nucleic acid detection kits, qRT-PCR kits are the mainstream, and the detection results are usually obtained within 1-2 hours, and the waiting time for detection is relatively long. [0004] Recombinase polymerase amplificatio...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2521/107C12Q2521/507C12Q2522/101C12Q2521/301C12Q2537/1376C12Q2565/625C12Q2563/107C12Q2545/113Y02A50/30
Inventor 李晓鲁蓝海
Owner CHENGDU HAIZHIYUAN BIOTECHNOLOGY CO LTD
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