Primer for rapidly detecting respiratory tract adenoviruses based on RAA fluorescent method, probe and detection kit

A detection kit and respiratory technology, applied in the field of molecular biology, can solve the problems of complex primer design, high false positive rate, and low sensitivity, and achieve the effects of convenient, rapid and accurate identification, high sensitivity, and reduced detection time

Inactive Publication Date: 2019-04-19
浙江国际旅行卫生保健中心 +1
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  • Abstract
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AI Technical Summary

Problems solved by technology

However, since cytopathy usually occurs within 2-7 days, this method takes a long period and is relatively cumbersome.
[0006] Immunofluorescence technology can be used not only for the identification of viruses in cell culture, but also for the detection of virus antigens in clinical specimens. It is fast, simple and has high specificity, but its sensitivity is low.
[0007] Molecular biology methods such as real-time fluorescent PCR have sensitivity and good characteristics, but require professional technicians, complex instruments and complete laboratories, and the time period also needs 1.5-2 hours; reverse transcription loop-mediated isothermal amplification method (LAMP) It has an isothermal temperature of 60-65°C and has good specificity, but the false positive rate is high and the primer design is complicated

Method used

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  • Primer for rapidly detecting respiratory tract adenoviruses based on RAA fluorescent method, probe and detection kit
  • Primer for rapidly detecting respiratory tract adenoviruses based on RAA fluorescent method, probe and detection kit
  • Primer for rapidly detecting respiratory tract adenoviruses based on RAA fluorescent method, probe and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Embodiment 1: Primer screening experiment

[0082] Upstream primers:

[0083] F1: 5'-CAGCATCAGTTTCACGAGCATCAACCTCTATGC-3';

[0084] F2: 5'-GCATCAGTTTCACGAGCATCAACCTCTATGCTA-3';

[0085] F3: 5'-CATCAGTTTCACGAGCATCAACCTCTATGCTAC-3';

[0086] Downstream primers:

[0087] R1: 5'-TGCGAGAAGGAATGGAAATGGGAATATTGGTTGC-3';

[0088] R2: 5'-CGAGAAGGAATGGAAATGGGAATATTGGTTGCAT-3';

[0089] R3: 5'-GAGAAGGAATGGAAATGGGAATATTGGTTGCATT-3';

[0090] The probe sequence is:

[0091] AGCCATGCTGCGGAATGACACCAATGATCAGTCATTCAACGACTAYCTATC

[0092] The probe is modified with a fluorescent reporter group (FAM) and a fluorescent quencher group (BHQ1);

[0093] The modified probe is

[0094] AGCCATGCTGCGGAATGACACCAATGATCAG(FAM-dT)C(THF)(BHQ1-dT)TCAACGACTAYCTATC;

[0095] The composition of the kit is shown in Table 1, and Table 1 is the composition list of the kit:

[0096] Table 1

[0097]

[0098] The prepared recombinant plasmid working standards are:

[0099] Working Standard 1, con...

Embodiment 2

[0107] Embodiment 2: Sensitivity experiment

[0108] Upstream primers:

[0109] 5'-CAGCATCAGTTTCACGAGCATCAACCTCTATGC-3'

[0110] Downstream primers:

[0111] 5'-TGCGAGAAGGAATGGAAATGGGAATATTGGTTGC-3'

[0112] The probe sequence is:

[0113] AGCCATGCTGCGGAATGACACCAATGATCAGTCATTCAACGACTAYCTATC

[0114] The probe is modified with a fluorescent reporter group (FAM) and a fluorescent quencher group (BHQ1);

[0115] The modified probe is:

[0116] AGCCATGCTGCGGAATGACACCAATGATCAG(FAM-dT)C(THF)(BHQ1-dT)TCAACGACTAYCTATC;

[0117] The composition of the kit is shown in Table 1.

[0118] The prepared recombinant plasmid working standards are:

[0119] Working Standard 1, containing 0.2 × 10 6 Copies / ul respiratory adenovirus plasmid non-infectious DNA fragment.

[0120] Working Standard 2, containing 0.2 x 10 5 Copies / ul respiratory adenovirus plasmid non-infectious DNA fragment.

[0121] Working Standard 3, containing 0.2 × 10 4 Copies / ul respiratory adenovirus plasmid non-i...

Embodiment 3

[0136] Embodiment 3: repeatability experiment

[0137] The sequences of the primer probe and the positive quality control product are the same as those in Example 1.

[0138] The composition of the kit is shown in Table 1.

[0139] The implementation method of the repeat experiment is:

[0140] (1) Reaction buffer preparation:

[0141] Draw 173 μL of reaction buffer from the reaction buffer tube in the kit and add it to the pre-prepared 1.5ml PE tube, then add 8 μL of the mixture of probe and primer (the concentration of the probe is 0.02mmol / , the concentration of the primer is 0.03mM) , and mix well to obtain the mixed reaction buffer.

[0142] (2) RAA fluorescent basic reaction reagent redissolved

[0143] Prepare 4 RAA fluorescent basic reaction reagents, each time draw 45 μL of the reaction buffer mixed in step 1 and add them to the prepared 4 RAA fluorescent basic reaction reagent tubes, so that the lyophilized powder is fully dissolved and mixed to become RAA React...

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Abstract

The invention provides a primer for rapidly detecting respiratory tract adenoviruses based on an RAA fluorescent method, a probe and a detection kit. The primer and the probe are suitable for RAA fluorescent method detection and can accurately detect the respiratory tract adenoviruses, the specificity can reach 100%, and the detection kit can conveniently, rapidly and accurately authenticate the respiratory tract adenoviruses. Operation is convenient, detection time is short, detection can be completed within 15 min, it is unnecessary to make DNAunqinding at the high temperature of 95 DEG C like PCR, then conduct annealing at the temperature of 50-60 DEG C and finally complete extending at the temperature of 72 DEG C, it is only needed to conduct isothermal amplification at the temperatureof 30-42 DEG C, and then detection is completed. It is also unnecessary to use 4-6 primers for amplification at the temperature of 65 DEG C like an LAMP technology, and false positive is not easily generated.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a primer, a probe and a detection kit for rapid detection of respiratory adenovirus based on RAA fluorescence method. Background technique [0002] Adenovirus is one of the most common viral pathogens in acute respiratory infections in children. The virus can be transmitted through people, water, media and equipment, and the period of existence in sewage can be extended to 3 weeks at room temperature. Adenovirus infection and large-scale epidemics are more likely to occur in children and military camp personnel, and most infants and young children have been infected with at least one adenovirus strain within 5 years after birth. Lipid sanitizers are ineffective and sensitive to heat, formaldehyde, and chlorine. Adenovirus infection has a global distribution and can occur year-round. Adenoviruses can infect the respiratory tract, intestinal tract, eyes, bl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2563/107
Inventor 郑伟王刚吕沁风杨天赐王玲吴忠华郭利川汤赛君王智宏应清界
Owner 浙江国际旅行卫生保健中心
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