Fluorescent RT-PCR detection reagent and method for coxsackievirus A16 and enterovirus 71

A technology of RT-PCR and coxsackie virus, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of reducing reagent detection performance, liquid reagents cannot adapt to the application of grassroots detection institutions, and reagent failure Risks and increased costs of cold chain management

Pending Publication Date: 2021-05-18
深圳市赛格诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, conventional fluorescent RT-PCR detection reagents are all liquid reagents, and there are strict cold chain requirements for transportation and storage (usually -20°C) to maintain reagent activity. will reduce the detection performance of the reagent
Once the traffic is blocked during long-distance transportation, the risk of reagent failure and the cost of cold chain management will increase greatly. It can be seen that liquid reagents cannot be used in long-distance transportation and grass-roots testing institutions with poor cold chain conditions.
[0007] In order to solve the many problems and inconvenience caused by the cold chain requirements of conventional liquid fluorescent RT-PCR detection reagents, a beneficial alternative is to make CA16 and EV71 fluorescent RT-PCR detection reagents into freeze-dried reagents that are stable at room temperature. At present, there is no report of CA16 and EV71 fluorescent freeze-dried RT-PCR detection reagents in the literature
At the same time, due to the increasing number of newly discovered nucleic acid sequences of CA16 and EV71, the fluorescent RT-PCR detection reagents designed based on the original nucleic acid sequence information face an increasing risk of missed detection

Method used

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  • Fluorescent RT-PCR detection reagent and method for coxsackievirus A16 and enterovirus 71
  • Fluorescent RT-PCR detection reagent and method for coxsackievirus A16 and enterovirus 71
  • Fluorescent RT-PCR detection reagent and method for coxsackievirus A16 and enterovirus 71

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. This example provides a design scheme of fluorescent RT-PCR primers and probes for detecting CA16 and EV71.

[0044] In the present invention, by analyzing the CA16 and EV71 nucleic acid sequence information in the public gene sequence database, selected figure 1 and figure 2 The nucleic acid sequences shown are respectively used as the detection target sequences of CA16 and EV71. Utilize the Primer Express V3.0 software to design the following primers for amplifying the target sequence and the probe sequences for detecting the target sequence:

[0045] The sequences of the specific forward primers of CA16 are SEQ ID No.1 (Tm=59.7°C) and SEQ ID No.2 (Tm=59.4°C), and the sequences of the reverse primers are SEQ ID No.3 (Tm=59.7°C ), the sequences of the probes are SEQ ID No.4 (Tm=68.8°C) and SEQ ID No.5 (Tm=69.6°C). The 5' end of the probe was modified with a fluorescent reporter group FAM, and the 3' end was modified with a fluorescent quencher group BHQ1...

Embodiment 2

[0049] Example 2: This example demonstrates that the CA16 and EV71 fluorescent RT-PCR detection reagents described in the present invention are made into freeze-dried reagents by vacuum freeze-drying technology.

[0050] Prepare CA16 and EV71 fluorescent RT-PCR liquid detection reagents according to the preferred components and contents described in Table 1, and divide them into 200 μl PCR eight-tube tubes according to 1 part / tube, then carry out vacuum freeze-drying, and the obtained CA16 and EV71 are freeze-dried The fluorescent RT-PCR detection reagent is white particles ( image 3 ). Add 23 μl of nuclease-free water to a reagent, shake slowly for 10 seconds, the reagent is completely dissolved, and the volume measured with a pipette gun is 25 μl, which shows that the volume of the reagent before reconstitution is equivalent to 2 μl. In order to prevent the reagents from getting wet and exposed, the CA16 and EV71 freeze-dried fluorescent RT-PCR detection reagents are first...

Embodiment 3

[0051] Example 3: This example demonstrates the detection effect of CA16 and EV71 nucleic acid positive reference products using the CA16 and EV71 freeze-dried fluorescent RT-PCR detection reagents of the present invention.

[0052] Carry out 10-fold serial dilution of the CA16 and EV71 nucleic acid positive reference substance mixture, mix 5 μl and 18 μl nuclease-free water for each concentration, and then add to 1 portion of CA16 and EV71 freeze-dried fluorescent RT-PCR prepared according to Example 2 In the detection reagent; 23 μl of nuclease-free water was used as a negative control. A reaction mixture with a volume of 25 μl was obtained, which was slowly oscillated to dissolve, centrifuged, and tested on a fluorescent PCR instrument. The reaction program was set according to Table 2. Each treatment was repeated 3 times.

[0053] Test results such as Figure 4 Shown, CA16 of the present invention and EV71 lyophilized fluorescent RT-PCR detection reagent are all positive...

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Abstract

The invention provides a fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) reagent and a method for detecting coxsackievirus A16 and enterovirus 71. A primer and a probe used by the fluorescent RT-PCR detection reagent for the coxsackievirus A16 and the enterovirus 71 provided by the invention are obtained by designing and screening based on the latest gene sequence information of the coxsackievirus A16 and the enterovirus 71 in a public gene sequence database, and have good inclusiveness, specificity and reaction performance; the reagent contains uracil DNA glycosylase and dUTP, so that a false positive result possibly caused by PCR product aerosol can be prevented, and the detection accuracy is further ensured; and besides, the fluorescent RT-PCR detection reagent for the coxsackievirus A16 and the enterovirus 71 is prepared into a freeze-dried reagent which can be stored at normal temperature through a freeze-drying technology, so that the risk that a conventional liquid fluorescent PCR detection reagent fails due to storage temperature change or repeated freeze-thawing is avoided, and the transportation and management cost is reduced.

Description

technical field [0001] The invention relates to pathogenic microorganism detection technology, in particular to the nucleic acid detection of coxsackievirus A16 and enterovirus 71. Background technique [0002] HFMD is a common infectious disease characterized by skin rashes or herpes, fever, sore throat, and general malaise on the hands, feet, and mouth. It was first reported in New Zealand in 1957. Hand, foot and mouth disease is caused by enterovirus infection, mainly through fecal-oral transmission, and can also be transmitted through contact with patient's feces, saliva and herpes fluid. People of different ages can be infected with the disease, mainly children under 5 years old, especially children under 3 years old with the highest incidence rate. [0003] Enteroviruses belong to the genus Enterovirus in the family Picornaviridae. Virus particles are spherical, with a diameter of 28-30 nm. The virus capsid is a three-dimensionally symmetrical icosahedron, composed of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2521/107
Inventor 黎绍基林镜中朱碧莹刘钰涵黄俊辉
Owner 深圳市赛格诺生物科技有限公司
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