Enterovirus triplex direct fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit and reaction system

An RT-PCR, enterovirus technology, applied in the field of molecular biology detection of enterovirus, can solve the problems of mutual interference between primers and probes, lack of RT-PCR, etc., to shorten detection time, improve detection efficiency, reduce The effect of the operation steps

Active Publication Date: 2016-04-13
深圳澳东医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the fact that primers and probes are easily affected by complex factors in the sample, and that primers and probes are prone to form complex structures and cause mutual interference, there are currently no suitable primers and probes that can be used to efficiently implement above purpose
[0006] In addition, there is currently a lack of suitable reaction solutions for the RT-PCR detection of triple direct amplification of enteroviruses universal, CA16, and EV71

Method used

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  • Enterovirus triplex direct fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit and reaction system
  • Enterovirus triplex direct fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit and reaction system
  • Enterovirus triplex direct fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit and reaction system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1 Kit of the present invention is used for the detection test of clinical sample

[0045] 1. Sample processing:

[0046] Pharyngeal swab samples: Use a sterile cotton swab to wipe the secretions from the palatine arches on both sides of the patient's mouth and pharynx and tonsils, avoiding touching other parts. Then quickly put the cotton swab into the collection tube provided by this kit, break the cotton swab rod near the top, add the internal standard solution, tighten the cap of the tube, mix well, and store it at 4°C for a short period of time.

[0047] 2. Reaction solution preparation:

[0048] In this embodiment, the concentration of Tris-HCl in the direct RT-PCR reaction solution is 31.25mmol / L, the concentration of ammonium sulfate in the direct RT-PCR reaction solution is 20.83mmol / L, and the concentration of potassium chloride in the direct RT-PCR reaction solution The concentration in the reaction solution is 72.92mmol / L, and the volume percenta...

Embodiment 2

[0060] Example 2 Comparison of analytical sensitivity between triple direct fluorescent RT-PCR and single conventional reagents for hand, foot and mouth

[0061] The nucleic acid sample of the CA16 standard strain of enterovirus was diluted to a nucleic acid concentration of 0.001LD50; according to the method and steps in Example 1, triple direct fluorescent RT-PCR was compared with single-fold routine reagent amplification detection, each concentration Repeat the detection 30 times. The results are shown in Table 3.

[0062] Table 3 Analytical Sensitivity Results

[0063]

[0064]

[0065] From the results shown in table 3, the analytical sensitivity of the direct fluorescent RT-PCR method of the present invention detects enterovirus is compared with the detection sensitivity of conventional reagent single-fold RT-PCR with purified RNA as a template, and this result illustrates that the direct fluorescent RT-PCR method The analytical sensitivity for detection of enter...

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Abstract

The invention discloses an enterovirus general type / CA16 / EV71 triplex direct fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit and a reaction system. The detection kit comprises primers and probe molecules of which the base sequences are disclosed as SEQ ID NO:1-9. The detection kit can simultaneously detect enterovirus general type, CA16 and EV71. By using the kit disclosed by the the invention, the detection can be performed after the sample is directly added into the reaction tube without the complex RNA (ribonucleic acid) purification step, thereby reducing the operation steps. The kit can be used for quickly and accurately detecting the enterovirus general type, shortens the detection time, enhances the detection efficiency, saves the cost, and can effectively lower the risk of cross contamination.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection of enterovirus, in particular to a detection kit and reaction system for triple direct fluorescent RT-PCR of enterovirus general type, CA16 type and EV71 type. Background technique [0002] Hand, foot, and mouth disease (HFMD) is an infectious disease caused by an enterovirus, which mostly occurs in preschool children, and can cause herpes on the hands, feet, and oral cavity of children. Pulmonary edema, aseptic meningoencephalitis and other complications, the mortality rate of severe children is high. The pathogens causing hand, foot and mouth disease are mainly Coxsackievirus (Coxasckievirus) A group 16, 4, 5, 7, 9, 10 types of Enterovirus, B group 2, 5, 13 types; , enterovirus general type and enterovirus 71 type (EV71 type), wherein EV71 type and CA16 type are the most common. [0003] At present, specific diagnostic techniques for enterovirus infection include five metho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 翟建新张利潘艳萍廖玉声杜维吴晓卫
Owner 深圳澳东医学科技有限公司
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