Magnetic capture method for SARS coronary virus and PCR detection therewith
A coronavirus and capture probe technology, applied in the field of capturing SARS coronavirus RNA, can solve problems such as the ability limitation of enrichment and purification
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Embodiment 1
[0116] Example 1: Capture and RT-PCR detection of standard RNA specimens without pre-hybridization
[0117] Take 50 μl of the standard SARS virus RNA obtained by the above method, add 100 μl of magnetic beads that have been bound to the capture probe P1, and incubate at room temperature for 90 minutes. Use a magnetic stand to collect and wash RNA, wash twice with B&W buffer, and wash twice with 1×PCR buffer, then dissolve RNA in 10μl DEPC water for reverse transcription and nested PCR amplification.
[0118] Reverse transcription of RNA:
[0119] Using M-MLV reverse transcriptase, take 10μl RNA sample for reverse transcription. The reaction system is:
[0120] 5×First strand cDNA buffer 4.0μl
[0121] 0.1M DTT 2.0μl
[0122] 10mM dNTP 1.0μl
[0123] Primer 2.5pmol
[0124] RNase inhibitor 40U
[0125] M-MLV 200U
[0126] RNA template 10μl
[0127] DEPC water to 20μl
[0128] The reaction conditions were 42°C, 30 min.
[0129]...
Embodiment 2
[0148] Example 2: Capture of pre-hybridized standard RNA specimens and RT-PCR detection
[0149] First, pre-hybridize 50μl standard RNA with 300pmol pre-hybridization probe PR1 in 6×SSPE and incubate at 54°C for 15min. Add 100μl of magnetic beads bound to the capture probe P1, and incubate for 90min at room temperature. Use a magnetic stand to collect and wash RNA, wash twice with B&W buffer, and wash twice with 1×PCR buffer, then dissolve RNA in 10μl DEPC water for reverse transcription and nested PCR amplification. The same reverse transcription and nested PCR amplification methods in Example 1 were used. The results of electrophoresis are as Figure 2B Shown.
Embodiment 3
[0150] Example 3: Capture of target RNA in culture medium of SARS coronavirus and RT-PCR detection
[0151] In the P3 laboratory, take 100μl of SARS coronavirus that has been successfully cultured and determined by TCID50, add 300μl TRIzol LS lysate, and after standing at room temperature for 5 minutes, add 80μl of chloroform, shake vigorously, and after standing at room temperature for 5 minutes, centrifuge at 12000×g at 4°C For 10 min, take 100 μl of the uppermost supernatant, add it to 6×SSPE containing 300 pmol of pre-hybridized probe PR1, and incubate at 54°C for 15 min. Add 10, 50, 100 μg of magnetic beads that have been bound to the specific probe P1 in Example 1, respectively. Then the reaction solution added with magnetic beads was incubated for 90 min at room temperature. Use a magnetic stand to collect and wash the RNA bound to the magnetic beads, wash twice with B&W buffer, and wash twice with 1×PCR buffer, then dissolve the RNA with magnetic beads in 10μl DEPC water a...
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