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Fulminating-infectious-disease pathogen detecting primer pair and kit

A pathogen detection and infectious disease technology, applied in the field of severe infectious disease pathogen detection, can solve the problems of inconvenient to carry large instruments, unfavorable detection, cumbersome operation of fluorescent quantitative PCR technology, etc., to shorten the detection time, reduce the sample size, and improve sensitivity and safety effects

Active Publication Date: 2016-04-13
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, fluorescent quantitative PCR technology is mainly used for detection, which has the advantages of fast and accurate detection results, but fluorescent quantitative PCR technology is relatively cumbersome to operate and needs to rely on large-scale instruments that are not easy to carry, which is not conducive to carrying out detection under field conditions

Method used

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  • Fulminating-infectious-disease pathogen detecting primer pair and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1: Specificity and sensitivity of Ebola primers

[0091] The specific components and dosage of the Ebola virus detection solution in the kit are shown in the following table:

[0092]

[0093] Take 1 μL of each sample, add it to the reaction solution, and block it. Reaction conditions: 63°C water bath for 1 hour. 85°C, 5min inactivation to stop the reaction, observe the color change, the result is as follows figure 1 As shown, in the detection solution containing Ebola primers, only the corresponding Ebola reaction pool turns sky blue ( figure 1 2) in , while the others are all violet.

[0094] Nephelometer detection: use the Qiagen kit to extract the nucleic acid in the above-mentioned Ebola plasmid mock sample, and dilute it according to a 10-fold gradient (V1:10 5 Copies / μL, V2: 10 4 Copies / μL, V3: 10 3 Copies / μL, V4: 10 2 Copies / μL, V5: 10 1 copies / μL, V6: 10 0 copy / μL, V7: 0 copy / μL, V8: negative control), the following amplification reaction sys...

Embodiment 2

[0096] Example 2: Specificity and sensitivity of Lhasa thermal primers

[0097] The specific components and dosage of the Lassa fever virus detection solution in the kit are shown in the following table:

[0098]

[0099]

[0100] The reaction solution in the microfluidic chip is all the above-mentioned Lassa fever virus detection solution.

[0101] Take 1 μL of the sample, add it to the reaction solution, and block it. Reaction conditions: 63°C water bath for 1 hour. 85°C, 5min inactivation to stop the reaction, observe the color change, the result is as follows Figure 4 As shown, in the detection solution containing the Sara heat primer, only the corresponding Sara heat reaction cell turns into sky blue ( Figure 4 3) in , while the others are all violet.

[0102]Nephelometer detection: Use the Qiagen kit to extract the nucleic acid in the Lhasa fever plasmid of the above-mentioned simulated sample, and dilute it according to a 10-fold gradient (V1:10 5 Copies / μL,...

Embodiment 3

[0103] Example 3: Marburg virus primer specificity and sensitivity

[0104] The specific components and dosage of the Marburg virus detection solution in the kit are shown in the following table:

[0105]

[0106]

[0107] The above-mentioned Marburg virus detection solution is used in the reaction pool of the microfluidic chip, and the detection method is the same as that in Example 1. The results are shown in Figure 7 , only the reaction pool corresponding to the Marburg virus template has a color change ( Figure 7 4) in , while the others are all violet.

[0108] Nephelometer detection: Use the Qiagen kit to extract the nucleic acid in the above-mentioned Marburg plasmid mock sample, and dilute it according to a 10-fold gradient (V1:10 5 Copies / μL, V2: 10 4 Copies / μL, V3: 10 3 Copies / μL, V4: 10 2 Copies / μL, V5: 10 1 copies / μL, V6: 10 0 copy / μL, V7: 0 copy / μL, V8: negative control), the following amplification reaction system was established: 1 μL nucleic acid...

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Abstract

The invention discloses a fulminating-infectious-disease pathogen detecting primer pair and a kit. The primer pair comprises at least a pair of RT-LAMP primers of Ebola viruses, Lassa fever viruses, Marburg viruses, rift valley fever viruses, yellow fever viruses and Chikungunya fever viruses. By means of the primer system, the amplification reaction background is reduced, and sensitivity and specificity are quite good. The kit formed by the primer pair further comprises detecting liquid and a micro-fluidic chip; as an independent RT-PCR secondary amplification step of a detecting liquid system is omitted, detecting time is shortened; as the denaturation process and the renaturation process of nucleic acid do not exist, the polluted chance of RNA enzymes and the polluted chance of amplification nucleic acid are reduced, and the sensibility and the safety of detection are improved. By means of the constant-temperature sealed environment provided by a micro-fluidic chip system, rapid and constant-temperature amplification and automation result distinguishing of a nucleic acid extracting template are finally achieved, the requirement for test hardware is reduced, the use level of a reaction reagent is reduced, detection cost is reduced, and the result can be directly determined through color changes.

Description

technical field [0001] The invention relates to the technical field of detecting pathogens of severe infectious diseases, in particular to a primer set and a kit for detecting pathogens of severe infectious diseases. Background technique [0002] In 2014, the worst Ebola epidemic in history broke out in West Africa, and it went out of Africa for the first time and spread to developed countries such as Europe and the United States, killing more than 11,000 people. The friendship between China and Africa has a long history, and the economic, trade and cultural exchanges are frequent and close. Therefore, the risk of Ebola epidemic importation is very high. After the outbreak, the Chinese government not only immediately provided a large amount of materials and technical assistance to African countries, but also dispatched medical and health experts to form a detection team and a medical team to help the local fight against the Ebola epidemic. [0003] Lassa fever (Lassafever) ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/70C12Q2565/629Y02A50/30
Inventor 郝荣章宋宏彬卢晓刘超贾雷立赵荣涛王瑞丽王金艳李杨孔文
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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