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A kit for simultaneous detection of 10 kinds of vector-borne disease pathogens and its application

A technology of insect-borne diseases and kits, applied in the field of liquid phase chip detection, to achieve the effect of optimized configuration, efficient coupling, and accurate detection

Active Publication Date: 2020-06-09
深圳澳东检验检测科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there is a method for detecting arboviruses by using a liquid-phase chip method, see Chinese patent CN102181532A, but the 9 kinds of arboviruses targeted by this method are only common arboviruses in my country, and it does not fully involve foreign epidemics. Insect-borne diseases, however, inspection and quarantine at foreign ports still need to carry out pathogen quarantine on animals or insects from countries with high incidence of insect-borne diseases, so as to improve my country's comprehensive prevention and control capabilities to prevent the long-distance spread of insect-borne foreign animal diseases, and to protect domestic animals. Not infected by foreign arbo-borne pathogens

Method used

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  • A kit for simultaneous detection of 10 kinds of vector-borne disease pathogens and its application
  • A kit for simultaneous detection of 10 kinds of vector-borne disease pathogens and its application
  • A kit for simultaneous detection of 10 kinds of vector-borne disease pathogens and its application

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Experimental program
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Effect test

Embodiment 1

[0052] The design of embodiment 1 primer and probe

[0053] Determine the specific fragments of each insect-borne disease by consulting OIE, national standards, industry standards and related literature, log in to GenBanK, and download the corresponding fragments of each insect-borne disease, namely WNV-E gene, VSV-N gene, ASFV-P72 Gene, BTV-NS1 gene, Borrelia burgdorferi-OSPA gene, EHDV-NS3 gene, Coxiella burnetii-IS1111a gene (accession number: M80806), SBV-S gene (accession number: HE649914), EBV-NP gene (accession number: KC545392 .1), RVFV-NSs gene (accession number: DQ380154). Using DNAMAN, MEGA, BioEdit, DNAStar, Primer Premier5.0 and other software to analyze and compare the gene sequences of the above 10 kinds of insect-borne disease pathogens, select the conserved regions, and after repeated studies and experiments, design, analyze and screen 10 Specific primers, 10 specific probes and 10 verification probes (reverse complement of probe, RC-Probe), labeling of the C...

Embodiment 2

[0064] Example 2 The optimization of the multiplex PCR detection system for simultaneously detecting 10 kinds of arbo-borne disease pathogens

[0065] 1. Extraction of pathogenic RNA of insect-borne diseases Use the QIAamp Viral RNAMini kit for RNA extraction, see the kit instructions for steps.

[0066] 2. Establishment of multiplex asymmetric PCR system

[0067] Optimize dNTP concentration, enzyme amount, annealing temperature and other conditions, especially pay attention to repeated optimization of the concentration ratio of upstream and downstream primers. Since the downstream primers are Biotin-labeled, the biotin-labeled single-stranded DNA is increased to improve hybridization efficiency.

[0068]In the asymmetric PCR reaction, the PCR reaction is carried out according to the concentration ratio of the upstream and downstream primers (1:1, 1:2, 1:3, 1:4, 1:5,), and then the hybridization detection is carried out to determine the suitable single multiple and the optim...

Embodiment 3

[0071] Embodiment 3 Preparation of liquid phase chip

[0072] 1. Coupling of probes and microspheres

[0073] According to the steps of probe microsphere coupling, WNV probe and #27 microsphere, BB probe and #28 microsphere, ASFV probe and #26 microsphere, VSV probe and #64 microsphere, BTV probe and #64 microsphere, BTV probe and #35 microsphere, Q Fever probe with #43 microsphere, RVFV probe with #47 microsphere, EBV probe with #21 microsphere, EHDV probe with #52 microsphere, SBV probe with #68 microsphere For coupling, the specific steps are as follows:

[0074] (1) First take out 2 fresh EDC powders and place them at room temperature (about 1 hour).

[0075] (2) Take out 10 kinds of microspheres of different colors (the concentration is 1.25×10 7 cells / mL) at room temperature, vortexed for 30s to suspend the microspheres and mix them thoroughly.

[0076] (3) Take 200 μL of the mixed microspheres into a 1.5ml brown EP tube, centrifuge at 14000g for 3-5min, put them int...

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Abstract

The invention provides a reagent kit for simultaneously detecting pathogens of ten vector-borne diseases and application of the reagent kit, and belongs to the technical field liquid chip detection. The reagent kit comprises specific primers for the pathogens of the ten common vector-borne diseases and probes coupled with fluorescence labeling micro-spheres. Nucleotide sequences of specific primer pairs are respectively shown as SEQ ID NO.1-20, and nucleotide sequences of the probes are shown as SEQ ID NO.21-30. The reagent kit and the application have the advantages that multiple asymmetric PCR (polymerase chain reaction) systems are set, hybridization reaction conditions are optimized, and accordingly the pathogens of the ten vector-borne diseases can be accurately detected; the reagent kit is accurate in detection and high in sensitivity and specificity, is simple, convenient and speedy, is suitable for inspection and quarantine in ports and epidemiological investigation on vector-borne diseases and has an excellent application prospect.

Description

technical field [0001] The invention relates to the technical field of liquid-phase chip detection, in particular to a method for simultaneously detecting 10 kinds of insect-borne disease pathogens by using a liquid-phase chip method, a detection kit and an application thereof. Background technique [0002] Insect-borne diseases refer to a disease that is transmitted from person to person, and from person to animal through the bite infection of blood-sucking arthropods. Clinical manifestations of animals include sudden fever, feeling sore and weak, extensive skin rash, and secondary infection leading to death. In the past, tens of thousands of humans and animals were infected or even died by the disease, which brought certain negative effects to the economic and human social development. After the middle of the 20th century, with the strengthening of global exchanges, it also promoted the continuous development of international animal trade, especially the use of modern tra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6834C12N15/11
CPCC12Q1/6834C12Q1/701C12Q2600/16C12Q2537/143C12Q2563/149Y02A50/30
Inventor 韩雪清王慧煜林祥梅梅琳肖荣海
Owner 深圳澳东检验检测科技有限公司
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