Dual-multienzyme-mediation-based nucleic acid amplification detection method and reagent
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A detection method and detection reagent technology, applied in the biological field
Pending Publication Date: 2021-03-16
苏州晶睿生物科技有限公司
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This method can achieve high-efficiency nucleic acid amplification at room temperature, but there is still room for optimization in its sensitivity and reaction efficiency.
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Embodiment 1
[0059] Example 1, Double multi-enzyme-mediated nucleic acid amplification detection of Shrimp Enterococcus hepatica
[0060] 1. Experimental reagents and equipment:
[0061] 1. Experimental reagents:
[0062] Harmless treated samples of Enterococcus hepatica;
[0064] The main components of the reagents for the detection of Shrimp Enterococcus hepatica by double multi-enzyme-mediated nucleic acid amplification are shown in Table 1:
[0065] Table 1
[0066]
[0067] Among them, diluent 1: every 100ml diluent contains 1M MgAc 22.80ml, 2M KAc 1.20ml, 1M Tris-Ac pH8.0 10.00ml, 30% PEG8K36.60ml, 0.1-1M dithiothreitol 0.40ml, water 49.00ml;
[0068] The composition of release solution 2 is as follows: every 100ml dilution contains 0.5M MgAc 2 2.80ml, 1.20ml of 1M KAc, 10.00ml of 0.5M Tris-Ac pH8.0, 36.60ml of 15% PEG8K, 0.40ml of 0.1-1M dithiothreitol, and 49....
Embodiment 2
[0098] Example 2, dEMA (Double Enzymes Mediated Amplification, double multi-enzyme-mediated nucleic acid amplification technology) detection of African swine fever
[0099] 1. Experimental reagents and equipment:
[0100] 1. Experimental reagents:
[0101] African swine fever negative and positive samples that have been harmlessly treated;
[0103] The main components of the reagents for the detection of African swine fever by double multi-enzyme-mediated nucleic acid amplification are shown in Table 4:
[0104] Table 4
[0105]
[0106] Among them, diluent 1: every 100ml diluent contains 1M MgAc 2 2.80ml, 2M KAc 1.20ml, 1M Tris-Ac pH8.0 10.00ml, 30% PEG8K 36.60ml, 0.1-1M dithiothreitol 0.40ml, water 49.00ml;
[0107] The composition of release solution 2 is as follows: every 100ml dilution contains 0.5M MgAc 2 2.80ml, 1.20ml of 1M KAc, 10.00ml of 0.5M Tri...
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Abstract
The invention relates to a dual-multienzyme-mediation-based nucleic acid amplification detection method. The method comprises the following steps: pre-amplification, freeze-dried redissolution, amplified detection and result analyzing. According to the method, a normal-temperature nucleic acid amplification technology is combined with a nest type amplification principle, a plurality of enzymatic reactions such as single-stranded DNA binding protein, DNA polymerase and DNA double-strand-opened enzyme are involved, DNA can be amplified by several million folds or more in 10 to 20 minutes under the constant-temperature condition of 30 DEG C to 45 DEG C, and rapid identification on the DNA can be achieved by being matched with a fluorescence detection technique. The method disclosed by the invention is simple in operation, short in time and low in instrument requirements and is applicable to rapid DNA diagnosis.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a double multi-enzyme-mediated nucleic acid amplification detection method and reagents. Background technique [0002] Nucleic acid amplification technology is divided into PCR technology and isothermal nucleic acid amplification technology, which correspond to the amplification of nucleic acid under temperature cycling and constant temperature respectively. In order to improve the efficiency of PCR amplification, nested PCR technology has been developed. Nested PCR is a variation of the polymerase chain reaction (PCR) that uses two (instead of one) pairs of PCR primers to amplify complete fragments. The amplified fragment of the first pair of PCR primers is similar to ordinary PCR, and the second pair of primers is called nested primers (because they are inside the fragment amplified by the first PCR) and binds inside the first PCR product, making the second PCR The amplified frag...
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