Design method for PCR primer, uses and reagent kit thereof

A design method and technology for designing primers, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as unsolvable problems, and achieve the effects of easy design, fewer synthetic primers, and simple composition.

Active Publication Date: 2009-01-14
SHANGHAI KOYEE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] The object of the present invention is to provide a kind of brand-new primer design method, the internal control line that utilizes this method to obtain is generally used in amplification detection hepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency virus (HIV) nucleic acid, to overcome traditional protein Detect inherent defects and deficiencies, and at the same time make up for many deficiencies of existing domestic and foreign similar products and even fatal flaws that cannot be solved methodologically

Method used

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  • Design method for PCR primer, uses and reagent kit thereof
  • Design method for PCR primer, uses and reagent kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The design of embodiment 1.HBV primer and probe, internal control probe:

[0033] 1.1. HBV primer: LBL-HBV01: 5'-aggaa cctct atgtt tccct-3'

[0034] LBL-HBV02: 5'-ccact cccat aggaa tcttg-3'

[0035] LBL-HBV probe01: 5'FAM-tgttg ctgta caaaa ccttc gga-NFQ3'

[0036] HBV sequence available for reference (the italic bold part is the primer binding sequence):

[0037] cttgttgctg tacaaaacct tcggacggaa actgcacttg

[0038] tattcccatc ccatcatcct gggctttcgc

[0039] 1.2. Internal control probe and sequence: LBL-HBV ic probe01: 5'ROX-tgttg ctgAT cTGaa ccttcgga-NFQ3'

[0040] Synthetic internal control sequence, the internal control sequence available for reference is (the part in italics is the primer binding sequence):

[0041] cttgttgctg c aacct tcggacggaa actgcacttg

[0042] tattcccatc ccatcatcct gggctttcg

[0043] Note: The uppercase bold base letters are designed mutation points

[0044] 1.3. Dilute the synthesized internal control sequence to 1000copies / ...

Embodiment 2

[0049] The design of embodiment 2.HCV primer and probe, internal control probe:

[0050] 2.1. HCV Primer: LBL-HCV01: 5'-cgtacagcct ccaggcc-3'

[0051] LBL-HCV02: 5'-gccgg gcata gagtg ggt-3'

[0052] LBL-HCV probe01: 5'FAM-cccct cccgg gagag ccata g-NFQ3'

[0053] HCV sequence available for reference (the part in italics is the primer binding sequence):

[0054] ccc ccctcccggg agagccatag tggtctgcgg aaccggtgag

[0055] tacaccggaa ttgccgggaa gactgggtcc tttcttggat aa

[0056] 2.2. Internal control probe and sequence: the internal control probe uses the same internal control probe as HBV, and the sequence is as follows:

[0057] LBL-HCV ic probe01: 5'ROX-tgttg ctgat ctgaa ccttc gga-NFQ3'

[0058] Synthesize the internal control sequence, the internal control sequence available for reference is:

[0059] cc tgttg ctgat ctgaa ccttc gga aaacccactc

[0060]

[0061] Note: The uppercase bold base letters are designed mutation points

[0062] 2.3. Dilute the synthesiz...

Embodiment 3

[0067] The design of embodiment 3.HIV primer and probe, internal control probe:

[0068] 3.1. HIV primer: LBL-HIV01: 5'-aggatgtata gccctattag-3'

[0069] LBL-HIV02: 5'-gaagc ttgct cggct ct-3'

[0070] LBL-HIV probe01: 5'FAM-ttctg gacat aaaac aaggg ccaaa aga-NFQ3'

[0071] Available HIV sequences for reference (the part in italics is the primer binding sequence):

[0072] cattctggac ataaaacaag ggccaaaaga atcctttaga

[0073] gactatgtag atcggttcta taaaactcta

[0074] 3.2. Internal control probe and sequence: the internal control probe uses the same internal control probe as HBV, and the sequence is as follows:

[0075] LBL-HIV ic probe01: 5'ROX-tgttg ctgat ctgaa ccttc gga-NFQ3'

[0076] Synthesize the internal control sequence, the internal control sequence available for reference is:

[0077] cc tgttg ctgat ctgaa ccttc gga aaacccactc

[0078]

[0079] Note: The uppercase bold base letters are designed mutation points

[0080] 3.3. Dilute the synthesized inte...

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Abstract

The invention provides a PCR primer design method and applications thereof and a kit, so as to overcome the intrinsic faults and defects of the traditional PCR detection and to make up for various defects in the existing products at home and abroad and even the inextricable fatal defects in methodology. According to the PCR primer design method, the annealing temperature of an internal control primer is lower than that of a target sequence primer, the target sequence is amplified and enriched under a high annealing temperature firstly, and then an internal control sequence is amplified under a low annealing temperature, so the suppression for the target sequence amplification can be avoided through controlling the amplification starting time of the internal control.

Description

Technical field: [0001] The invention belongs to the field of nucleic acid diagnostic reagents, and specifically relates to a brand-new, efficient and easy-to-control method for designing PCR primers, and using the method to design multiple PCR detection kits for nucleic acid of hepatitis and AIDS involving internal control, and the kits produced by the method High sensitivity, very stable and reliable internal control, suitable for clinical nucleic acid testing and blood nucleic acid screening. Background technique: [0002] PCR technology (polymerase chain reaction technology) is an in vitro enzymatic reaction that specifically amplifies DNA. It can amplify DNA between two known sequences in a short time for diagnosis, identification, preparation of probes and genes. Engineering product development, etc., is an extremely effective and practical technique. Due to certain false positive and false negative problems in PCR tests, the application of PCR technology in clinical ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/70
Inventor 李刚
Owner SHANGHAI KOYEE BIOTECH CO LTD
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