PCR primer set detecting severe acute respiratory syndrome (SARS)-coronavirus, method and kit for detecting sars-coronavirus using the same
a technology of coronavirus and primer set, which is applied in the field of pcr primer set for detecting severe acute respiratory syndrome (sars) virus, a method and a kit for detecting sars virus, which can solve the problems of false negative or false positiv
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example 1
Design of Primers for SARS-CoV Detection
[0050]In this Example, there were designed PCR primers for SARS-COV detection, which are specific to SARS-CoVs and do not react with other coronaviruses.
[0051]The genome sequences of 40 strains belonging to SARS-CoVs were searched using the public database (www.ncbi.nim.nih.gov). Based on the search results, common sequences of the 40 strains were extracted. After sequence homology search between the common sequences and other coronaviruses and other respiratory viruses capable of infecting human bodies, homologous sequences were removed and sequences with no cross reactivity were selected. The results are presented in Table 1 below.
TABLE 1Analysis results for cross reactivity using B12Seq or BlastnH_CoVHumanSectionH_CoV229EOC43INFVPINFVRSVADVHMPVgDNASequenceNS*NSNSNSNSNSNSPM**matching*NS: No significant homology**PM: Partially matchingH_CoV229E: Human coronavirus 229E,H_CoV OC43: Human coronavirus OC43INFV: Influenza virus type A, B, PINFV: P...
example 2
Tube RT-PCR for SARS-CoV RNA Samples
[0064]In this Example, RT-PCR was performed on a PCR machine (ABI 2400) using the primer sets of GS-SARS01-06 and SARS-CoV RNA (total RNA samples extracted from SARS-CoVs) as templates. At this time, the concentration of the templates was 2 μg / 20 μl. Before initiating PCR, the RNA samples in a 25 μl PCR solution were converted to cDNAs by reverse transcription at 42° C. for 60 minutes. The PCR was performed in three-step thermal cycling conditions as follows: 50 cycles of denaturation at 95° C. for 20 seconds, annealing 65° C. for 40 seconds, and extension at 72° C. for 60 seconds.
[0065]The results are shown in FIG. 2. As shown in FIG. 2, SARS-CoV RNAs were detected. In FIG. 2, lane 1 is 1 kb-DNA plus ladder (Invitrogen), lane 2 is a PCR product (156 bp) amplified with GS-SARS01, lane 3 is a PCR product (244 bp) amplified with GS-SARS02, lane 4 is a PCR product (179 bp) amplified with GS-SARS05, lane 5 is a PCR product (103 bp) amplified with GS-S...
example 3
RT-PCR for SARS-CoV RNA Samples on Silicon-Based PCR Chip
[0066]In this Example, RT-PCR was performed on a PCR machine (GenSpector 1&2) using the primer sets of GS-SARS01-06 and SARS-COV RNAs (total RNA samples extracted from SARS-CoVs) as templates. At this time, the concentration of the templates was 2.4×103 copies. Before initiating PCR, the RNA samples in a 1 μl PCR solution on a silicon-based PCR chip were converted to cDNAs by reverse transcription at 42° C. for 60 minutes. The PCR was performed in three-step thermal cycling conditions as follows: 50 cycles of denaturation at 92° C. for 1 second, annealing 52° C. for 1 second, and extension at 72° C. for 5 seconds.
[0067]The results are shown in FIG. 3. As shown in FIG. 3, SARS-COV RNAs were detected on the silicon-based PCR chip. Each lane of FIG. 3 is as defined in FIG. 2.
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