Novel high-sensitivity respiratory virus nucleic acid NASBA (nucleic acid sequence based amplification) primers and detection method

An amplification primer and high-sensitivity technology, applied in the field of biomedicine, can solve the problems of increased pollution opportunities, long time-consuming, multiple scientific research purposes, etc., and achieve the effects of improving RNA amplification efficiency, improving detection sensitivity, and saving operation time.

Active Publication Date: 2016-01-20
HANGZHOU JOINSTAR BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method needs to first reverse-transcribe the viral RNA into cDNA, and then perform the amplification reaction of the specific nucleic acid sequence, which increases the chance of contamination and limits the wide application of this technology in clinical testing.
The fourth category is gene chips, next-generation sequencing technology, and mass spectrometry. Although these advanced detection technologies have greatly improved sensitivity, they are time-consuming or expensive, and h...

Method used

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  • Novel high-sensitivity respiratory virus nucleic acid NASBA (nucleic acid sequence based amplification) primers and detection method
  • Novel high-sensitivity respiratory virus nucleic acid NASBA (nucleic acid sequence based amplification) primers and detection method
  • Novel high-sensitivity respiratory virus nucleic acid NASBA (nucleic acid sequence based amplification) primers and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Reagent composition (taking respiratory syncytial virus as an example)

[0082] (1) Primer

[0083] FP: AATTCTAATACGACTCACTATAGGGAGAAGGAAAGTCCTACAAAAAAATGC

[0084] RP: AATTCTAATACGACTCACTATAGGGAGAAGGATCTATCTCCTGCTGCTAAT 0.25ul each.

[0085] (2) NASBA Amplification Reagent

[0086] BA (buffer): 2ul

[0087] DA:dNTP0.25ul

[0088] NA: NTP0.5ul

[0089] DMSO: 0.25ul

[0090] EM (enzyme mix): AMV, RNAseH, T7 RNA polymerase, BSA5.5ul

[0091] (3) NASBA product detection reagent

[0092] 1% agarose gel

Embodiment 2

[0093] Embodiment 2: NASBA detection process

[0094] (1) Amplification system: Add 1ul sample RNA template, 2ulBA, 0.25ulNA, 0.5ulDA, 0.25ulDMSO, 0.25ulFP, 0.25ulRP into a 0.2ml PCR amplification tube.

[0095] (2) Amplification process: Place the PCR amplification tube on the amplification instrument, heat it at 65° for 5 minutes, then cool it at 42° for 5 minutes; then add 5.5ulEM, and react at 42° for 60 minutes.

[0096] (3) Amplification product detection: Take 2ul of sample buffer and add it to the amplification product to terminate the reaction.

[0097] The amplified product was added to 1% agarose gel, and electrophoresis standard was added to one of the sample wells. After electrophoresis at 100V for 10 minutes, the presence or absence of the target band was observed in the electrophoresis imager.

Embodiment 3

[0098] Embodiment 3: comparative experiment

[0099] Adopt the contrast of respiratory syncytial virus NASBA kit of the present invention and one-step PCR method to dilute the secondary standard of respiratory syncytial virus into 10 2 、10 4 -10 10 copy / ul. Use the kit of the present invention and one-step PCR to detect simultaneously.

[0100] (1) NASBA kit sensitivity test

[0101] NASBA amplification and detection were performed according to Example 2.

[0102] From attached figure 2 It can be seen that the NASBA kit of the present invention can successfully detect 10 2 Copy the sample of / ul.

[0103] (2) One-step PCR sensitivity and rigidity detection

[0104] A. One-step PCR reaction system: 2ul sample RNA template, 12.5ul2*one-stepSYBRRT-PCRbuffer, 1ulprimeScript1stepEnzymeMix, 1ulFP, 1ulrp, 7.5ulRNaseFreeddh 2 0.

[0105] B. One-step PCR reaction process: put the above PCR tube at 42° for 5 minutes and then enter the cycle without going, 95° for 10 seconds,...

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Abstract

The invention provides novel high-sensitivity respiratory virus nucleic acid NASBA (nucleic acid sequence based amplification) primers. A respiratory virus conservative area is selected, and a promoter sequence capable of being recognized by T7 RNA (ribose nucleic acid) polymerase is formed at the 5' terminal of one of the synthesized primers. The invention further provides a novel high-sensitivity respiratory virus nucleic acid NASBA detection method. T7 RNA polymerase promoter sequences are added to the 5' terminals of the two primers complementary to two ends of a respiratory virus RNA sequence, so that when the primers enter a circulation phase, RNA sequences [RNA(+)] consistent with a template sequence can be synthesized, RNA sequences [RNA(-)] complementary to a template RNA sequence can be synthesized, and the detection sensitivity of viral nucleic acid is greatly improved; a throat swab sample of a patient can be directly used for amplification, a reverse transcription process is not required, and the operation time is saved.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a gene amplification technology for respiratory virus detection, and more specifically to establish a high-sensitivity NASBA amplification technology for respiratory virus nucleic acid. Specifically, it includes a pair of respiratory virus-specific amplification primers, a NASBA amplification reaction system and a NASBA product detection system. Background technique [0002] Acute respiratory infection is one of the main causes of human morbidity and death. There are many types of pathogens that cause infection, including bacteria, viruses, mycoplasma and chlamydia, etc., especially viruses. Regardless of the etiology, the clinical symptoms and signs of respiratory tract infections are similar, while infections caused by different types of pathogens have completely different treatment methods, and the curative effects and course of the disease are also different. [0003] R...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6865C12Q1/701C12Q2531/143
Inventor 郑昭璟朱友杰耿娟李桃萍赵艳任杰张冉朱叶孙晋华周旭一
Owner HANGZHOU JOINSTAR BIOTECH
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