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Quintuple fluorescent PCR (polymerase chain reaction) rapid hypersensitive detection kit for Ebola and application thereof

A fluorescent detection and re-detection technology, applied in the direction of recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., can solve difficult problems

Active Publication Date: 2014-12-17
苏州华益美生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Partly because of the danger of research, and partly because of preventing information from falling into the hands of bioterrorists, the genetic background of Ebola virus (including the complete genome sequence, etc.) is not fully disclosed to the public, especially Ebola -EBO-Zaire, EBO-Sudan, EBO-Bundibugyo, EBO-Taiforest ) and Ebola-Reston type (EBO-Restor) virus genome homology between the five subtypes of the virus, making it necessary to design five non-interfering specific primers for these virus subtypes pair and probes, especially difficult

Method used

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  • Quintuple fluorescent PCR (polymerase chain reaction) rapid hypersensitive detection kit for Ebola and application thereof
  • Quintuple fluorescent PCR (polymerase chain reaction) rapid hypersensitive detection kit for Ebola and application thereof
  • Quintuple fluorescent PCR (polymerase chain reaction) rapid hypersensitive detection kit for Ebola and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0087] Example 1 Extraction of Nucleic Acid

[0088] Under the license and supervision of the Clinical Inspection Center of the Ministry of Health, the nucleic acids of the following Ebola virus standard samples were extracted: Ebola-Zaire type (EBO-Zaire), Ebola-Sudan type (EBO- Sudan), EBO-Bundibugyo, EBO-Taiforest and EBO-Restor.

[0089] The extraction of nucleic acid is carried out according to the magnetic bead extraction method. In order to adapt to the nucleic acid extraction of the above five viruses at the same time, the improvement is as follows: add 90uL nucleic acid extraction solution to 1uL standard products of various dilutions (the formula and final concentration are: isothiocyanate Guanidine 1.2M, sodium edetate (pH8.0) 10mM, Tween-202% (W / W), sodium perchlorate 1M, ethanol 40% (V / V), Tris-HCl (pH8. 0)10mM), incubate at 42°C for 10min, then add 10uL D-Beads DNA magnetic bead suspension (50mg / mL, can be purchased from Beijing Aibigen Biotechnology Co., Ltd....

Embodiment 2

[0090] Example 2 Five-fold real-time PCR test

[0091] 1. Primer and probe sequences

[0092] Commission the synthesis of the following primer pairs and probes:

[0093] Primer pairs for detection of Ebola-Zaire:

[0094] EBV-Z-F:GCCACTCACGGACAATGACA,

[0095] EBV-Z-R:GCATGCGAGGGCTGGTT,

[0096] Probes to detect EBO-Zaire:

[0097] EBV-Z-P:AAGAAATGAACCCTCCGGC,

[0098] Primer pairs for detection of Ebola-Sudan (EBO-Sudan):

[0099] EBV-S-F: GGCAGCTCTTGGCTCACTTG,

[0100] EBV-S-R: TGAGGAGACGTGCAAATGGA,

[0101] Probes to detect Ebola-Sudan (EBO-Sudan):

[0102] EBV-S-P:CAAGCATGGAGAATAT,

[0103] Primer pairs for detection of Ebola-Bundibugyo:

[0104] EBV-B-F: ACCTCCGACGCGGTACAC,

[0105] EBV-B-R: TGTGGGTGAACAGGAACATCA,

[0106] Probes to detect Ebola-Bundibugyo:

[0107] EBV-B-P:CAAGACAGGCAACCTA,

[0108] Primer pairs for detection of Ebola-Taiforest:

[0109] EBV-T-F: GACCCGGATGATGGCAGAT,

[0110] EBV-T-R:GGCATTCGCCGTCTCACTA,

[0111] Probes to detect Ebola-T...

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Abstract

The invention provides a real-time PCR (polymerase chain reaction) method for performing quintuple detection on target nucleic acid in a nucleic acid extracting solution in a single PCR reaction container, which is used for simultaneously detecting whether EBO-Zaire, EBO-Sudan, EBO-Bundibugyo, EBO-Taiforest, EBO-Restor and other Ebola virus subtypes exist. The method can effectively avoid production of false negative and false positive results, and is of great practical significance for Ebola virus detection. The invention also provides a detection kit for implementing the method, application thereof and the like.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection. Specifically, the invention relates to a multiplex real-time PCR method capable of rapidly detecting multiple types of Ebola virus at the same time, which can quickly, sensitively and complete the detection of Ebola-Zaire type ( EBO-Zaire), EBO-Sudan, EBO-Bundibugyo, EBO-Taiforest and EBO- Reston type (EBO-Restor) and other five kinds of disease-causing pathogen detection. In addition, the present invention also relates to the reagents involved in the above PCR method, such as primers, probes, etc. that are irrelevant to each other, as well as the detection kit used in the above method and the preparation and application of the corresponding detection kit. Background technique [0002] Ebola virus (Ebola virus) is a virus that seriously threatens human life, and its biosafety level is 4 (while AIDS and SARS are only 3). The Ebola virus is mainly transmitted through the blood and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/70C12Q1/701C12Q2600/16C12Q2531/113C12Q2537/143C12Q2561/113C12Q2563/107
Inventor 刘劲林郭志武陈红干张必新王川李振勇
Owner 苏州华益美生物科技有限公司