Chikungunya virus testing method
A chikungunya virus and primer sequence technology, applied in the detection/testing of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc., to achieve important social and economic benefits
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Embodiment 1
[0031] Embodiment 1, design and synthesis of primers and probes
[0032] The SeqMan program in the Lasergene software package was used to analyze the nucleotide sequences of 37 strains of Chikungunya virus obtained from Genbank, select non-structural protein genes with highly conserved nucleotides, and use Primer Express2.0 software to design specific primers and The probe design, primers and probe sequences are listed in Table 1.
[0033] Table 1
[0034]
[0035] The location of the primers and probes on the Chikungunya virus genome was based on the strain sequence with the GenBank sequence number AF490259.
Embodiment 2
[0036] Example 2. Preparation of positive control RNA template and extraction of viral RNA for specificity assessment
[0037] According to the sequence of Chikungunya virus (GenBank sequence number: AF490259), synthesize a DNA fragment with a size of 120bp including the fragment sequence detected by fluorescent RT-PCR, clone it on the T vector (TOPO TA cloning kit), and use plasmid DNA extraction reagent The plasmid DNA was purified by the cassette, and after sequencing confirmed that the inserted fragment was correct, reverse transcription was performed in vitro: first, linearize the plasmid with Not I to exclude downstream RNA products, then perform in vitro transcription with MAXIscript T7Kit to generate target RNA, and then use Turbo DNase Perform DNase treatment to remove untranscribed plasmid DNA templates, and finally use ethanol precipitation to purify the RNA product, which is the positive control RNA template, and store it in a -70°C ultra-low temperature refrigerato...
Embodiment 3
[0040] Embodiment 3, determination of amplification program
[0041] The kit used for TaqMan RT-PCR reaction is Ag-Path-ID TM One step RT-PCR Kit (product of Ambion, USA), amplification and detection were carried out on Applied Biosystems 7500Fast Real-Time PCR Systems, a fully automatic fluorescent quantitative instrument, or on instruments such as Stratagene Mx3005P real-time fluorescent quantitative PCR instrument.
[0042]According to the recommendations of the kit and the specific characteristics of the primers and probes, the reaction program on the instrument tested 3 temperatures and 3 times in the selection of annealing extension conditions: the reverse transcription reaction conditions were 50°C for 10 minutes, and 95°C hot start for 10 minutes. Minutes later, enter 45 two-step PCR cycles: 95°C for 10 seconds → (58 / 60 / 62)°C (30 / 40 / 60) seconds; ×RT-PCR buffer 10 μl, 20 μM forward primer CHIK-FP 1 μl, 20 μM reverse primer CHIK-RP 1 μl, 5 μM probe CHIK-Probe 0.5 μl, 2...
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