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Internal reference quality control primer group for detecting aquatic pathogens by LAMP method and application of primer group

A technology of quality control primers and primer sets, applied in the direction of microorganisms, recombinant DNA technology, and methods based on microorganisms

Pending Publication Date: 2020-12-01
FISHERIES RES INST OF FUJIAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Since the LAMP reaction is easily affected by factors such as detection reagents (such as reaction enzyme inactivation), the quality of nucleic acid extraction of the amplified template (such as the extracted nucleic acid is severely degraded or the nucleic acid is not extracted due to operational errors), therefore, setting up the LAMP reaction The internal reference quality control of the detection is particularly important; at present, the reports on the internal reference quality control are mostly found in human disease detection, but there are few reports on the internal reference quality control of the detection of aquatic pathogens
Moreover, most of the existing internal reference quality control primer sets are only aimed at the same organism, and there are few reports on internal reference quality control primer sets suitable for multiple organisms.

Method used

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  • Internal reference quality control primer group for detecting aquatic pathogens by LAMP method and application of primer group
  • Internal reference quality control primer group for detecting aquatic pathogens by LAMP method and application of primer group
  • Internal reference quality control primer group for detecting aquatic pathogens by LAMP method and application of primer group

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] The gene sequence of the 18S ribosomal RNA of multiple species such as shrimps, crabs, fish and ginseng is compared with the 18S ribosomal RNA gene sequence of Litopenaeus vannamei shown in SEQ IDNo:1; when using When there are many aligned sequences, it is impossible to obtain sequences with highly conserved regions; therefore, Macrobrachium rosenbergii18S (GQ131934), Penaeus indicus 18S (MH400902), Penaeus semisulcatus 18S (DQ079766), Procambarus clarkii 18S (AF436001), Scylla Paramamosain 18S (KC902763) was multiple-aligned with the sequence shown in SEQ ID No: 1; the highly conserved region of the sequence shown in SEQ ID No: 1 was obtained by screening; according to the conserved region, PrimerExplorer V4 software was used to design the internal reference quality control LAMP primer set; The invention designed a total of four sets of primers, which are respectively marked as NCZK-1, NCZK-2, NCZK-3, and NCZK-4. Each set of primers contains two inner primers (FIP and ...

Embodiment 2 4

[0072] Embodiment 2 Amplification analysis of four sets of internal reference quality control primer sets

[0073] Template preparation: Extract Procambarus clarkii, Australian freshwater lobster, blue crab, large yellow croaker, puffer puffer, oblique banded grouper, gentian grouper, Litopenaeus vannamei, rough sea cucumber, sea cucumber, Genomic DNA of 11 species such as sea cucumber was imitated, and used for amplification analysis of four sets of primers NCZK-1, NCZK-2, NCZK-3, and NCZK-4.

[0074] 1. The NCZK-1, NCZK-2, NCZK-3, and NCZK-4 primer combinations are respectively based on the genomic DNA of Procambarus clarkii, Australian freshwater lobster, blue crab, large yellow croaker, and Litopenaeus vannamei, and Water was used as a negative control, and the LAMP reaction was carried out at 63° C. for 60 minutes. After the reaction, the amplification result was analyzed by observing the amplification curve.

[0075] The LAMP reaction system (25 μL) is:

[0076]

[...

Embodiment 3

[0110] A kit for detecting common pathogenic microorganisms in prawns by LAMP method, including: internal reference quality control primer set and IHHNV virus primer set, white spot syndrome virus primer set, hepatic enterocystosis primer set, prawn iridescent virus primer set, prawn acute hepatopancreatic necrosis At least one of the primer sets for vibrio parahaemolyticus; also contains reaction buffer, Bst DNA polymerase, dNTPs, ddH 2 O (RNase-free) and a chromogenic dye; wherein, the chromogenic dye is any one of calcein, HNB, and phenol red reagent.

[0111] The IHHNV virus primer set sequence is:

[0112] F3: 5'-AACCCTCCACCAGACAAGA-3' SEQ ID No: 26 B3: 5'-TGTAGACATCTGTGTGGGTCT-3' SEQ ID No: 27 FIP: 5'-CGGCGCACATGGTTGTCTATGATCACCAGCGACGACTTCCT-3' SEQ ID No: 28 BIP: 5'-TTCAACAAGAGCAAGCCCAAGGACTTGATCCTTCGGCGTGTT-3' SEQ ID No: 29 LF: 5'-CTTTTCGTATTCTTGGAAGAGTCCT-3' SEQ ID No: 30 LB: 5'-GGAGGGATCCACATAATGAAGACG-3' SEQ ID No: 31 ...

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Abstract

The invention discloses an internal reference quality control primer group for detecting aquatic pathogens by an LAMP method and application of the internal reference quality control primer group, andbelongs to the technical field of development of aquatic pathogen detection reagents. The internal reference quality control primer group for detecting the aquatic pathogen by the LAMP method is designed according to a nucleic acid sequence shown in SEQ ID No: 1 or a complementary base sequence or a transcribed RNA sequence of the nucleic acid sequence. The internal reference quality control primer group disclosed by the invention can be used for detecting pathogens of various aquatic animals such as fishes, shrimps, crabs, sea cucumbers and the like, and lays a foundation for construction ofa pathogen detection system of the various aquatic animals.

Description

technical field [0001] The invention belongs to the technical field of developing reagents for detecting aquatic pathogens, and in particular relates to an internal reference quality control primer set for detecting aquatic pathogens by a LAMP method and an application thereof. Background technique [0002] my country is one of the major aquatic product production and export countries in the world, and aquaculture is the main pillar industry of the agricultural economy. In recent years, with the development of the aquaculture industry and the continuous expansion of the aquaculture scale, the occurrence of aquaculture diseases has become more frequent and the damage has become more and more serious, which is one of the most important factors restricting the development of the entire aquaculture industry. According to incomplete statistics, from 2002 to 2009, the average annual loss of aquaculture diseases in my country reached 10 billion yuan, of which fish accounted for abo...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6893C12Q1/689C12Q1/6844C12N15/11C12R1/63
CPCC12Q1/701C12Q1/689C12Q1/6893C12Q1/6844C12Q2600/166C12Q2531/119Y02A40/81
Inventor 葛辉周宸林琪吴建绍
Owner FISHERIES RES INST OF FUJIAN