RT-LAMP detection primer pairs, kit and detection method for infectious haematopoietic necrosis virus (IHNV)
A technology of RT-LAMP and hematopoietic organ necrosis, applied in the field of molecular biology, can solve the problems of long detection period, not adapting to the requirements of the times, etc., and achieve the effects of simple operation, high sensitivity and simple identification.
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Embodiment 1
[0040] Example 1 Establishment of Infectious Hematopoietic Necrosis Virus RT-LAMP Detection Kit
[0041] RT-LAMP detection kit for infectious hematopoietic necrosis virus, including RT-LAMP primer set, RT-LAMP reaction solution, Bst DNA polymerase, AMVReverse transcriptase, positive and negative controls.
[0042] (1) RT-LAMP primer design: using the envelope glycoprotein gene of infectious hematopoietic necrosis virus (IHNV) ( glyG Gene, GenBank accession number is DQ164100.1) as the target gene, using the online design software PrimerExplorerversion4 (http: / / primerexplorer.jp / e) to design LAMP primers. The primer sequences are listed in Table 1.
[0043] Table 1 Primer sequence list
[0044]
[0045] (2) RT-LAMP reaction solution: containing 10mMdNTP, 10×ThermoPol reaction buffer, and 150mMMgSO4 aqueous solution, the volume ratio of the three is 8:5:2.
[0046] (3) The positive control is a T vector clone containing the envelope glycoprotein gene fragment of infe...
Embodiment 2
[0048] Example 2 Using a turbidimeter to establish an infectious hematopoietic necrosis virus RT-LAMP detection method:
[0049] Utilize the method for detecting infectious hematopoietic necrosis virus with the kit of embodiment 1, comprise the steps:
[0050] (1) Extraction of RNA from samples to be tested: Extract RNA from samples using a virus RNA extraction kit;
[0051] (2) Constant temperature gene amplification reaction: 25μl reaction system contains: IHNV-F30.2μM, IHNV-B30.2μM, IHNV-FIP1.6μM, IHNV-BIP1.6μM, IHNV-LF0.8μM, IHNV-LB0.8μM, RT-LAMP reaction solution 12.5μl, AMV reverse transcriptase 1U, Bst 8U of DNA polymerase, 1~100ng of RNA to be tested, make up to 25μl with DEPC water; set up positive control and negative control; mix the prepared PCR tube and centrifuge, and react at 63~65℃ for 60~90min, and in 80°C for 2 minutes;
[0052] (3) Result judgment: judge the amplification result by observing the turbidity change of the precipitate in the reaction tube...
Embodiment 3
[0053] Example 3 ESE-Quanttube scanner detection
[0054] The kit is the same as in Example 1 except that the chromogenic agent (SYBRGreenI) not in Example 1 is added.
[0055] Use the above kit to detect infectious hematopoietic necrosis virus (IHNV) by the following method:
[0056] (1) Extraction of RNA from the sample to be tested: Extract and purify the RNA from the sample to be tested using a viral RNA extraction kit;
[0057] (2) Constant temperature gene amplification reaction: 25μl reaction system contains: IHNV-F30.2μM, IHNV-B30.2μM, IHNV-FIP1.6μM, IHNV-BIP1.6μM, IHNV-LF0.8μM, IHNV-LB0.8μM, RT-LAMP reaction solution 12.5μl, AMV reverse transcriptase 1U, Bst DNA polymerase 8U, 10×SYBRGreenI0.5μl, RNA to be tested 1~100ng, make up to 25μl with DEPC water; set positive control and negative control; mix the prepared PCR tube and centrifuge, react at 65℃ for 60min, and Continue at 80°C for 2 minutes;
[0058] (3) Result judgment: Place the reaction tube in ESE-Q...
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