Primer set for use in detection of enteroviruses by loop-mediated isothermal amplification technique and detection system
A loop-mediated constant temperature, technical detection technology, applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc. It is easy to popularize and apply on a large scale, has broad market prospects, and is easy to identify.
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Embodiment 1
[0044] Example 1: Specific Genome Sequence Search and LAMP Primer Design
[0045] Enterovirus genome sequences were retrieved from GenBank, and homology analysis was performed by BLAST software to find the specific conserved target sequences of enteroviruses.
[0046] LAMP primer design
[0047] The designed specific LAMP primer set (primers for detection of enterovirus with loop-mediated constant temperature amplification technique) includes primers as follows:
[0048] sequence
[0049] After comparison and analysis in the GenBank database, the sequences with high similarity to this group of enterovirus universal primers are derived from various enteroviruses, including coxsackievirus, echovirus, enterovirus 71 and other new enteroviruses. Viruses, no other microbial sequences with high similarity were found (Table 1).
[0050] Table 1: Some sequences highly similar to enterovirus universal primers
[0051] AY421760.1
[0052] FJ198212.1
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Embodiment 2
[0054] Embodiment 2: the establishment of detection system
[0055] By setting different final concentrations of Mg 2+ (2mmol / L, 3mmol / L, 4mmol / L, 5mmol / L, 6mmol / L, 7mmol / L), dNTP (1.2mmol / L, 1.4mmol / L, 1.6mmol / L, 1.8mmol / L, 2.0mmol / L), Betaine (0mol / L, 0.2mol / L, 0.4mol / L, 0.6mol / L, 0.8mol / L, 1mol / L, 1.2mol / L) and temperature (60℃, 61℃, 62℃ , 63°C, 64°C, 65°C), and optimized to obtain the best reaction parameters, so as to establish an enterovirus LAMP detection system. One of the optimized detection systems (20μL) is as follows:
[0056] 2.5 μL of 10× ThermoPol buffer;
[0057] 25mmol / L Mg2+ 2μL;
[0058] 5mol / L Betaine 4μL;
[0059] 4.5 μL of 10 mmol / L dNTP;
[0060] 0.5 μL of the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing of 10 μmol / L;
[0061] 0.5 μL of the nucleotide sequence shown in SEQ ID NO.2 in the sequence listing of 10 μmol / L;
[0062] 0.4 μL of the nucleotide sequence shown in SEQ ID NO.3 in the sequence listing of 100 μmol / L;
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Embodiment 3
[0068] Embodiment 3: A detection system for detecting enteroviruses using loop-mediated constant temperature amplification technology, the system is composed of the following raw materials:
[0069] 2.5 μL of 10× ThermoPol buffer;
[0070] 25mmol / L Mg 2+ 1 μL;
[0071] 5mol / L Betaine 3.5μL;
[0072] 5 μL of 10 mmol / L dNTP;
[0073] 0.5 μL of the nucleotide sequence shown in SEQ ID NO.1 in the sequence table of 10 μmol / L;
[0074] 0.5 μL of the nucleotide sequence shown in the sequence listing SEQ ID NO.2 of 10 μmol / L;
[0075] 0.4 μL of the nucleotide sequence shown in the sequence listing SEQ ID NO.3 of 100 μmol / L;
[0076] 0.4 μL of the nucleotide sequence shown in the sequence listing SEQ ID NO.4 of 100 μmol / L;
[0077] 1 μL of 8U / μL Bst enzyme;
[0078] Add ultrapure water to a total volume of 20 μL.
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