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Triple detection method of arbovirus liquid chips

A technology of arboviruses and arboviruses, applied in the direction of microorganism-based methods, resistance to vector-borne diseases, biochemical equipment and methods, etc., can solve problems such as difficult distinction and identification, simplify operation steps and reduce workload , The effect of improving the sensitivity

Inactive Publication Date: 2014-10-29
李云峰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the clinical manifestations of the above three viral diseases are similar, and they are mainly prevalent in Africa, Asia and other regions, and it is difficult to distinguish and differentiate through simple body temperature monitoring and epidemiological case investigation

Method used

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  • Triple detection method of arbovirus liquid chips
  • Triple detection method of arbovirus liquid chips
  • Triple detection method of arbovirus liquid chips

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1: PCR multiple amplification

[0072] 1) Design of primers and probes

[0073] Primer 5.0 software was used to design primers and probes, both of which were synthesized by Songbao Bioengineering (Dalian) Co., Ltd., and the sequences and primer-related information are shown in Table 1;

[0074] Table 1 Liquid-phase chip detection genes and related primers and probe sequences

[0075]

[0076] In addition, we also used the online sequence alignment tool http: / / multalin.toulouse.inra.fr / multalin / multalin.html to perform multiple sequence alignments on the three probes, and the results showed that the sequence consistency of the three probes was poor , indicating that the probability of the three probes forming a cross-reaction is very small, and the results are as follows figure 2 shown;

[0077] Since the simultaneous amplification of multiple primers is involved in the multiplex PCR amplification system, the performance requirements of the primers f...

Embodiment 2

[0091] Embodiment 2: Preparation of microsphere mixture

[0092] 1) Nucleic acid probes are coupled to fluorescently encoded microspheres

[0093] The principle of the coupling reaction between carboxylated fluorescently encoded microspheres and aminated probes is as follows: image 3 shown. The three nucleic acid probes designed in this research are coupled with different numbers of fluorescently encoded microspheres, and the numbers of nucleic acid probes and corresponding fluorescently encoded microspheres are shown in the table below;

[0094] Table 5 Nucleic acid probes and microsphere numbers

[0095]

[0096] The coupling method of nucleic acid probes and fluorescently encoded microspheres is operated according to the instruction manual of the nucleic acid liquid phase chip coupling kit provided by Shenzhen Borui Xianghui Biotechnology Co., Ltd. The specific method is as follows:

[0097] 2) Experiment preparation:

[0098] Equilibrate all reagents to room tem...

Embodiment 3

[0128] Example 3: Establishment of arbovirus liquid chip triple detection method, hybridization of PCR product and microsphere mixed solution, liquid chip detection

[0129] In the hybridization reaction, four gradients are set for the loading amount of PCR products, which are 1 μL / reaction, 3 μL / reaction, 5 μL / reaction, and 7 μL / reaction, and the optimal loading amount is determined according to the test results;

[0130] 1) Experimental steps

[0131] Shake the resuspended microsphere mixture at full speed for about 3 minutes;

[0132] For each sample well and background well, add 10 μL of microsphere mixture, and then add 33 μL of nucleic acid detection buffer 1 (1.5×TMAC);

[0133] For each background well, add 1μL / 3μL / 5μL / 7μL PCR blank product;

[0134] For each sample well, add 1μL / 3μL / 5μL / 7μL of PCR product;

[0135] Make up the volume to 50 μL with Nucleic Acid Detection Buffer 2 (1.0×TMAC);

[0136] Thoroughly mix the components in the reaction well;

[0137...

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Abstract

The invention discloses a triple detection method of arbovirus liquid chips. The method comprises the following steps: respectively designing specific primers and probes aiming at three types of arboviruses comprising Chikungunya viruses, Crimean-Congo hemorrhagic fever and Rift Valley fever viruses, determining the specificity of the primers and the probes via a single PCR and clone sequencing, and then carrying out multiplex-PCR amplification under the condition that the concentration ratio of the forward and reverse primers in an amplification reaction system is 1:1-1:8; then coupling the probes with fluorescent coding microspheres so as to prepare a microsphere mixing liquid and carrying out coupling quality control by coupling the probes; finally, carrying out a hybridization reaction on multiplex-PCR products and the microsphere mixing liquid, simultaneously adding streptomycin-phycoerythrin diluted by a nucleic acid detection buffer solution, and analyzing reaction products by utilizing a liquid chip detecting instrument. The method is good in detection specificity and can be used for simultaneously detecting the three types of arboviruses, and has high detection sensitivity due to the adoption of an asymmetric PCR amplification method in the process of the multiplex-PCR amplification.

Description

technical field [0001] The invention relates to a liquid phase chip triple detection method capable of simultaneously detecting three arboviruses of chikungunya fever virus, Crimea-Congo hemorrhagic fever virus and Rift Valley fever virus and the preparation of the involved liquid phase chip methods and applications. Background technique [0002] Arboviruses refer to a class of viruses that use blood-sucking insects as the medium to transmit a variety of serious diseases among vertebrates, humans and animals. At present, about 537 species of arboviruses have been discovered, distributed all over the five continents of the world, and more than 130 species have been confirmed to be pathogenic to humans and animals [1] . In virus classification, arboviruses belong to 14 virus families. According to the types of viruses included and their relationship with human and animal diseases, arboviruses are mainly concentrated in the genus Alphavirus (29 species) of the family Togavir...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6816C12Q1/70C12Q2537/143C12Q2563/149C12Q2531/107Y02A50/30
Inventor 李云峰蔡鹏龙川凤赵亮葛藤
Owner 李云峰
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