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Enterovirus real-time fluorescent quantitative detection kit

A technology of real-time fluorescence quantification and detection kit, applied in the field of diagnosis

Inactive Publication Date: 2016-01-13
FAPON BIOTECH INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] No enterovirus real-time fluorescent quantitative detection kit for distinguishing EV71 type enterovirus from CA16 type enterovirus has been reported yet

Method used

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  • Enterovirus real-time fluorescent quantitative detection kit
  • Enterovirus real-time fluorescent quantitative detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0064] A real-time fluorescent quantitative detection kit for enteroviruses is provided, which is used to distinguish EV71 type enteroviruses from CA16 type enteroviruses.

[0065] This enterovirus real-time fluorescent quantitative detection kit includes: upstream primer EV71F1 for amplifying EV71 enterovirus, downstream primer EV71R1 for amplifying EV71 enterovirus, and EV71 enterovirus detection Probe EV71P1, the two ends of the probe EV71P1 are respectively combined with a first fluorescent group and a first fluorescent quencher; the upstream primer CA16F2 for amplifying CA16 type enterovirus is used for amplifying CA16 type enterovirus The downstream primer CA16R2 of the virus is used to detect the probe CA16P2 of CA16 enterovirus. The two ends of the probe CA16P2 are respectively combined with a second fluorescent group and a second fluorescent quencher. The first fluorescent group and the second fluorescent The groups are different; the upstream primer F3 used to amplif...

Embodiment 2

[0068] Embodiment 2 The minimum detection amount of the kit

[0069] A commercial nucleic acid RNA extraction kit OMEGAE.Z.N.A.TotalRNAKitI (OMEGA, R6834-01) was used to extract viral RNA from virus isolation cultures to obtain samples.

[0070] The concentration of nucleic acid in the sample was measured with a UV-visible photometer, and the viral nucleic acid in the sample was sequentially diluted to 1.0×10 with sterile deionized water. 6 copy, 1.0×10 5 copy, 1.0×10 4 copy, 1.0×10 3 copy, 1.0×10 2 copy, 10 copies and 1 copy.

[0071] After detection and quantification, the minimum detection amount of the enterovirus real-time fluorescence quantitative detection kit is 10 copies.

Embodiment 3

[0072] Accuracy and specificity of embodiment 3 kits

[0073] The isolated cultures of EV71 enterovirus and CA16 enterovirus were mixed as the experimental group, and hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV) and human cytomegalovirus were selected. (HCMV), Mycoplasma pneumoniae (MP) and Treponema pallidum (TP) isolated cultures were mixed as a control group.

[0074] Among them, the selected hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), Mycoplasma pneumoniae (MP) and Treponema pallidum (TP) were tested by corresponding reagents. All the cassettes were positive and confirmed by sequencing as corresponding pathogenic microorganisms.

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Abstract

The invention discloses an enterovirus real-time fluorescent quantitative detection kit, which is used for distinguishing EV71 enterovirus from CA16 enterovirus, wherein the detection kit comprises a forward primer EV71F1 and a reverse primer EV71R1 for amplifying the EV71 enterovirus, a probe EV71P1 for detecting the EV71 enterovirus, a forward primer CA16F2 and a reverse primer CA16R2 for amplifying the CA16 enterovirus, a probe CA16P2 for detecting the CA16 enterovirus and a real-time fluorescent quantitative PCR (polymerase chain reaction) buffer solution, wherein a first fluorescence group and a first fluorescence quencher are respectively bonded to two ends of the probe EV71P1; a second fluorescence group and a second fluorescence quencher are respectively bonded to two ends of the probe CA16P2; and the first fluorescence group is different from the second fluorescence group. The enterovirus real-time fluorescent quantitative detection kit, when being used, can distinguish the EV71 enterovirus from the CA16 enterovirus by respectively detecting signals of the first fluorescence group and signals of the second fluorescence group.

Description

technical field [0001] The invention relates to the technical field of diagnosis, in particular to a real-time fluorescence quantitative detection kit for enterovirus. Background technique [0002] HFMD is an infectious disease caused by enteroviruses. There are more than 20 enteroviruses (types) causing HFMD, among which Enterovirus 71 (EV71) and Coxsackievirus A16 (CoxA16, CA16 ) is the most common. The HFMD caused by the two is clinically indistinguishable. Unlike the CA16 type, the EV71 type can not only cause HFMD, but more importantly, it can cause severe central nervous complications, such as encephalitis, meningitis, Acute flaccid paralysis, etc., even lead to death. At present, there is a lack of effective treatment drugs mainly for symptomatic treatment. [0003] The incubation period of hand, foot and mouth disease, viral encephalitis and other diseases caused by enterovirus is generally 3 days to 7 days, without obvious prodromal symptoms, and most patients sh...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q2531/113C12Q2561/113C12Q2563/107
Inventor 杨浩邓艳华胡鹏
Owner FAPON BIOTECH INC
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