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

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

Example Embodiment

[0034] Embodiment 1 is used to detect the synthesis of the LAMP primer set of Brucella

[0035] The Brucella-specific conserved gene-insertion sequence IS711 was selected in GenBank as the target gene (DQ845343.1 in GenBank). According to the sequence of positions 141080~141282, the online tool http: / / primerexplorer. jp / e / V4 designs, screens and synthesizes a set of primers whose parameters meet the requirements of LAMP primer design. These include 2 inner primers (FIP, BIP), 2 outer primers (F3, B3) and 2 loop primers (LB, LF). The sequence is shown in Table 1:

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

[0037]

Example Embodiment

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

[0039] 1.1 Preparation of standards

[0040] Amplify the DNA of the standard strain of Brucella ovum 16M (purchased from the Brucella strain storage bank of the Institute of Infectious Diseases, Chinese Center for Disease Control and Prevention) with the outer primers (F3, B3), and recover the target fragment after cutting the gel. It was connected to the PUCm18-T vector (purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.) to prepare a plasmid standard.

[0041] 1.2 Optimization of LAMP test parameters

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

[0043] 1.3Mg 2+ Concen...

Example Embodiment

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

[0060] The plasmid standards diluted to different concentration gradients were added to the optimized reaction system, and then LAMP reaction was carried out. The products were electrophoresed on a 2.0% agarose gel and imaged, and the highest dilution of the plasmid with the target band appeared as the sensitivity. The evaluation index to determine the lower limit of detection of the LAMP method.

[0061] The 10-fold serial dilution of the Brucella plasmid DNA standard was tested by LAMP and conventional PCR respectively. The results showed that LAMP could detect the Brucella plasmid standard of 10fg level at least, which was the highest detection sensitivity (100fg level) of conventional PCR. 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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