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LAMP primer for detecting Brucella and kit containing the same

A technology of Brucella and LAMP-PCR, which is applied in the detection field of Brucella, can solve the problems of large investment, high requirements for instruments and equipment, and barriers to popularization of technology at the grassroots level

Inactive Publication Date: 2012-12-12
ICDC CHINA CDC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the conventional PCR method for Brucella detection requires high equipment (such as PCR instrument, gel imaging system) and requires a lot of investment, which has caused great obstacles to the promotion of the technology at the grassroots level.

Method used

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  • LAMP primer for detecting Brucella and kit containing the same
  • LAMP primer for detecting Brucella and kit containing the same
  • LAMP primer for detecting Brucella and kit containing the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 is used for detecting the synthesis of the LAMP primer set of Brucella

[0035] Select Brucella specific conserved gene in GenBank—insert sequence IS711 as the target gene (number DQ845343.1 in GenBank), and use the online tool http: / / primerexplorer according to the sequence of 141080-141282. jp / e / V4 designed, screened and synthesized a set of primers whose parameters met the requirements of LAMP primer design. These include 2 inner primers (FIP, BIP), 2 outer primers (F3, B3) and 2 loop primers (LB, LF). See Table 1 for the sequence:

[0036] Table 1 Primer sequence of Brucella LAMP detection method

[0037]

Embodiment 2

[0038] Construction of embodiment 2LAMP reaction system and optimization of reaction parameters

[0039] 1.1 Preparation of standard products

[0040] Use external primers (F3, B3) to amplify the DNA of the standard strain 16M of Brucella melis (purchased from the Brucella Species Preservation Bank of the Institute of Infectious Diseases, China Center for Disease Control and Prevention), and extract the target fragment after gel cutting. Connected to the PUCm18-T vector (purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.) to make a plasmid standard.

[0041] 1.2 Optimization of LAMP test parameters

[0042] Using the positive plasmid standard DNA as a template, the Mg in the system was 2+ The optimal LAMP reaction parameters were determined after optimizing the concentration, ratio of internal and external primers, Bst enzyme concentration, dNTP concentration, betaine concentration, reaction temperature and reaction time.

[0043] 1.3Mg 2+ Concentra...

Embodiment 3

[0059] Embodiment 3 utilizes LAMP primer set to detect the sensitivity analysis of Brucella

[0060] Plasmid standards diluted into different concentration gradients were added to the optimized reaction system, and then LAMP reaction was performed. The product was electrophoresed and imaged on a 2.0% agarose gel. The highest dilution of the plasmid with the target band was used as the sensitivity To determine the lower limit of detection of the LAMP method.

[0061] The 10-fold serially diluted Brucella plasmid DNA standard was tested by LAMP and conventional PCR respectively. The results showed that LAMP could detect the lowest 10fg level of the Brucella plasmid standard, which was higher than the conventional PCR detection sensitivity (100fg level). 10 times( figure 2 ).

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Abstract

The invention provides an LAMP primer for detecting Brucella and a kit containing the same. The primer comprises 2 inner primers ( FIP, BIP ), 2 outer primers ( F3, B3 ) and 2 loop primers ( LB, LF ) (shown as Seq ID No.1-6). According to the invention, the LAMP primer isothermal amplification technology is applied to the rapid detection of Brucella, so as to fast, accurately and conveniently detect the Brucella in a serum specimen. The method has higher specificity and sensitivity than a conventional PCR method, and has vital significance to early prevention and pathogenic monitoring of Brucella; at the same time, the invention can avoid high equipment investment, and is more suitable for Brucella epidemic survey site or local health and epidemic prevention.

Description

technical field [0001] The invention relates to the detection of Brucella, in particular to a LAMP primer for detecting Brucella and a kit containing the primer. Background technique [0002] Brucella is a gram-negative intracellular parasite with no spores and no flagella and is pathogenic to humans and animals. It is the pathogen of brucellosis (brucellosis). Traditional Brucella species include sheep (3 types), cattle (8 types), pigs (5 types), dogs (1 type), sheep epididymis (1 type) and Sandrat species (1 type), a total of 6 species and 19 types. There are three types of Brucella that infect humans. Patients with brucellosis have symptoms such as fever, hyperhidrosis, hepatosplenomegaly, joint and general muscle pain; misdiagnosis and mistreatment of the etiology of brucellosis tend to turn into a chronic phase, and the chronic phase of brucellosis has a long course of disease and is prone to relapse or reinfection, which can lead to Labor force is reduced or even lo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 姜海崔步云张利田国忠朴东日赵鸿雁
Owner ICDC CHINA CDC
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