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Title:
DIRECTIONAL POLYMERISATION FLUORESCENT PROBE PCR AND TEST KIT
Document Type and Number:
WIPO Patent Application WO/2019/228541
Kind Code:
A1
Abstract:
A directional polymerisation fluorescent probe PCR and a test kit, wherein directional primer pair 5' end complementary binding, sensitising, fluorescent probe 3' end directional polymerisation and melting PCR are performed on the basis of primers and probe specific sequences, primer design uses pair side original primer 5' end 5-10bp reverse base sequences, added to primer pair front ends according to the 5'-3' direction to form a "5' reverse complementary sequence" chimeric primer pair for directional polymerisation, and 5' complementarity enables amplification product 3' ends to also be mutual templates and mutual primers for amplification. Sensitivity is increased by >2n geometric progression amplification, and competition decreases primer 3' polymerisation PD non-specificity. Conventional hydrolysis fluorescent probe 3' ends are added to probe central portion reverse complementary 5-8bp end sequences to form two probe molecules for end portion-central portion close mutual hybridisation and directional polymerisation. Fluorescein-quenching groups at both ends of the probes are close to reduce reaction baseline fluorescence, and probe self-polymerisation inhibits primer polymerisation non-specificity. Once long sequence target molecule chains compete for hybrid primers and probe chains, polymerisation primer pairs and probes with weak relative binding forces melt and bind to target template molecules for amplification, and probes are hydrolysed by polymerase to produce fluorescence.

Inventors:
JIANG HONG (CN)
QU YUE (CN)
QU CHAOQI (CN)
Application Number:
PCT/CN2019/094327
Publication Date:
December 05, 2019
Filing Date:
July 02, 2019
Export Citation:
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Assignee:
JIANG HONG (CN)
International Classes:
C12Q1/6876
Domestic Patent References:
WO2016146968A12016-09-22
WO2010105074A12010-09-16
Foreign References:
CN108774639A2018-11-09
CN103114131A2013-05-22
CN105400886A2016-03-16
Attorney, Agent or Firm:
BEIJING HAIHONG JIACHENG INTELLECTUAL PROPERTY & PARTNERS (CN)
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Next Patent: PRIMER 5' END REVERSE COMPLEMENTARY FLUORESCENT PCR AND TEST KIT
2n geometric progression amplification, and competition decreases primer 3' polymerisation PD non-specificity. Conventional hydrolysis fluorescent probe 3' ends are added to probe central portion reverse complementary 5-8bp end sequences to form two probe molecules for end portion-central portion close mutual hybridisation and directional polymerisation. Fluorescein-quenching groups at both ends of the probes are close to reduce reaction baseline fluorescence, and probe self-polymerisation inhibits primer polymerisation non-specificity. Once long sequence target molecule chains compete for hybrid primers and probe chains, polymerisation primer pairs and probes with weak relative binding forces melt and bind to target template molecules for amplification, and probes are hydrolysed by polymerase to produce fluorescence."/>