CRISPR-Cas12b-based ultra-sensitive rapid detection and identification system for hepatitis B virus type B and hepatitis C virus type C
A hepatitis B virus, sensitive technology, applied in the field of ultra-sensitive rapid detection and identification system, can solve difficult problems such as hepatitis B virus typing and detection
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Embodiment 1
[0048] 1. Preparation of target DNA and clinical samples
[0049] In this study, the full-length DNA sequence of the S gene of type B HBV was synthesized (see Genbank Accession No.AF100309 for the sequence of the S gene of type B HBV), and the full-length DNA sequence of the S gene of type C HBV (type C The sequence of the S gene of HBV is shown in Genbank Accession No.AB014381). The S gene of type B HBV and the S gene of type C HBV were respectively integrated into the pUC57 vector using conventional means of the prior art to obtain a type B plasmid and a type C plasmid (General Biol Company, Anhui, China). At the same time, the full-length DNA sequences of the S gene of A, D, E, F, G and H HBV types were also obtained (Genbank Accession No.: AF090842, X65259, AB032431, AB036910, AF160501 and AY090454), And use pUC57 vector to construct A-type plasmid, B-type plasmid, D-type plasmid, E-type plasmid, F-type plasmid, G-type plasmid and H-type plasmid. 114 serum samples of sus...
experiment example 1
[0067] Experimental Example 1: Study on Sensitivity
[0068] Using B-type plasmid and C-type plasmid as templates, the sensitivity of MCDA-CRISPR-real-time fluorescence detection and MCDA-CRISPR-LFB was studied, and the amount of B-type plasmid and C-type plasmid was 1×10 5 ,1×10 4 ,1×10 3 ,1×10 2 ,1×10 1 ,1×10 0 ,1×10 -1 Copies each reaction and set up blank control (BC) at the same time. Experimental results such as Figure 8 as shown, Figure 8 In A and B, the B-type plasmid is used as a template, Figure 8 In C and D, use the C-type plasmid as a template, Figure 8 A and C show MCDA-CRISPR-LFB, Figure 8 B and D show MCDA-CRISPR-real-time fluorescence detection, Figure 8 "+" means positive, "-" means negative. It can be seen from the experimental results that the LOD (the limited of detection) of MCDA-CRISPR-real-time fluorescence detection and MCDA-CRISPR-LFB is 10copies per reaction. In this experimental example, for MCDA-CRISPR-LFB, other parameter conditi...
experiment example 2
[0069] Experimental Example 2: Research on Optimal Reaction Conditions
[0070] Use B-type plasmid and C-type plasmid as template (1.0×10 3 copies each reaction), the temperature conditions of MCDA amplification were studied (60-67°C), and real-time monitoring was carried out using a turbidimeter. For the experimental results, see Figure 9 with Figure 10 , the results showed that 65°C was the optimum reaction temperature.
[0071] The cutting time of CRISPR-Cas12b was studied, and the reaction time was selected as 1, 5, 10 and 20 min for research. For experimental results, see Figure 11 , Figure 11 In A and C, the type B plasmid is used as a template, Figure 11 In B and D, the type C plasmid is used as the template, Figure 11 A and B show MCDA-CRISPR-LFB, Figure 11 C and D show MCDA-CRISPR-real-time fluorescence detection. The experimental results show that for MCDA-CRISPR-LFB, CRISPR-Cas12b can be cut for 2 minutes (type B) and 5 minutes (type C), and the detec...
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