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LAMP primer composition and kit for detecting swine acute diarrhea syndrome coronavirus by using microfluidic chip technology

A technology of microfluidic chip and primer composition, which is applied in the field of nucleic acid detection and diagnosis, and can solve the problems of laborious, time-consuming and high cost of coronavirus detection

Inactive Publication Date: 2020-04-10
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In summary, the combination of microfluidic chip technology and LAMP detection method can establish a specific, stable, sensitive, fast and efficient instant detection system, which has important application value in the detection of porcine acute diarrhea syndrome coronavirus , which is expected to solve the time-consuming, laborious and high-cost defects of porcine acute diarrhea syndrome coronavirus detection

Method used

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  • LAMP primer composition and kit for detecting swine acute diarrhea syndrome coronavirus by using microfluidic chip technology
  • LAMP primer composition and kit for detecting swine acute diarrhea syndrome coronavirus by using microfluidic chip technology
  • LAMP primer composition and kit for detecting swine acute diarrhea syndrome coronavirus by using microfluidic chip technology

Examples

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Embodiment 1

[0038] Example 1 Preparation of LAMP primer composition for detection of porcine acute diarrhea syndrome coronavirus by microfluidic chip technology

[0039] The design and synthesis steps of the LAMP primer composition are as follows:

[0040] First, retrieve the full-length genome sequences of all SADS-CoV from the Genbank database, and perform homology analysis by BLAST software to find the relatively conserved target gene (M gene) sequence of the SADS-CoV genome. According to the obtained target gene sequence, use PrimerExplorer V5 software designed LAMP primer composition and synthesized primers;

[0041] Then, the synthesized primers are screened, that is, the synthesized primers are dissolved and the primers are screened, and finally the specific and sensitive primer composition required for the microfluidic chip is obtained. The primer composition is composed of outer primer F3 / B3, inner primer FIP / BIP and loop primer LB / LF, wherein F3 has the nucleotide sequence show...

Embodiment 2

[0048] Embodiment 2 Structure of microfluidic chip

[0049] Such as figure 1 As shown, the microfluidic chip used in the present invention is a disc-shaped microfluidic chip, which is produced by Shanghai Suchuang Diagnostic Products Co., Ltd., and its model is 8×4, which includes 8 reaction detection areas 1, Each reaction detection zone 1 comprises a sampling pool 2, a distribution pool 5, a capillary microvalve 4, and an amplification pool 3 connected in sequence, the sampling pool 2 communicates with the distribution pool 5 through an arc channel, and the sampling pool 2 is provided with There is a sampling hole, and the distribution pool 5 communicates with the waste liquid pool and the exhaust hole through an arc channel; each reaction detection area 1 is equipped with four amplification pools 3 .

[0050] Among them, the main function of the sampling pool 2 is to load the reaction solution; the main function of the distribution pool 5 is to evenly distribute the reacti...

Embodiment 3

[0051] Example 3 Establishment of a kit for detecting porcine acute diarrhea syndrome coronavirus using microfluidic chip technology

[0052] The kit involved in the present invention includes the LAMP primer composition for detecting porcine acute diarrhea syndrome coronavirus described in Example 1, the microfluidic chip described in Example 2, and also includes a constant temperature amplification premix, Reverse transcriptase solution, RNase inhibitor.

[0053] In the above-mentioned LAMP primer composition for detecting porcine acute diarrhea syndrome coronavirus, the molar ratio between the outer primer F3 / B3, the inner primer FIP / BIP and the loop primer LB / LF is 1:8:4, the described The concentration of each primer component in the LAMP primer composition was 50 μM.

[0054] The above isothermal amplification premix is ​​a reaction solution containing 800 U / mL of Bst DNA polymerase and 50 μM fluorescent dye SYBRGreen (provided by Shanghai Suchuang Diagnostic Products C...

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Abstract

The invention belongs to the field of nucleic acid detection and diagnosis, and in particular relates to a loop-mediated isothermal amplification (LAMP) primer composition and kit for detecting a swine acute diarrhea syndrome coronavirus (SADS-CoV) by using a microfluidic chip technology. In order to solve the defect of time-consuming, laborious and high-cost detection of a swine acute diarrhea syndrome coronavirus, LAMP primers are designed according to a relatively conservative M gene of the SADS-CoV, after screening is performed, the LAMP primer composition for detecting the swine acute diarrhea syndrome coronavirus is obtained. The invention also provides the kit combining the microfluidic chip technology and an LAMP technology and a detection method of the SADS-CoV by using the kit. The composition and kit are applied to detection of the swine acute diarrhea syndrome coronavirus, and can realize the rapid and immediate detection of the swine acute diarrhea syndrome coronavirus, thereby solving the defect of time-consuming, laborious and high-cost detection of the swine acute diarrhea syndrome coronavirus; and the composition and kit improve the detection sensitivity and specificity, reduce labor and equipment costs, and shorten the detection cycle.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection and diagnosis, and in particular relates to a LAMP primer composition and a kit for detecting porcine acute diarrhea syndrome coronavirus using microfluidic chip technology. Background technique [0002] Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel coronavirus that can cause severe swine acute diarrhea syndrome (SADS). SADS-CoV mainly causes watery diarrhea, severe vomiting, emaciation and dehydration in piglets. The mortality rate of infected piglets under 5 days old is as high as 90%, and the mortality rate of infected piglets over 8 days old is less than 5%. The study found that SADS-CoV is not related to the three coronaviruses currently circulating in pig farms, such as porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus, PEDV), porcine deltacoronavirus (Porcine Deltacoronavirus, PDCoV), porcine transmissible gastroenteritis Antibodies to the virus (Porci...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2565/629C12Q2537/1376C12Q2521/107C12Q2563/107C12Q2527/127Y02A50/30
Inventor 蓝天周玲陈咏辉卢先东方雪恩刘艳红孔继烈
Owner SOUTH CHINA AGRI UNIV
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