Kit for detecting brucella and use method thereof

A detection kit, the technology of Brucella, applied in the field of biological detection reagents, can solve the problems of many required reagents, long detection period, easy cross-contamination operation process, etc.

Active Publication Date: 2012-10-10
GUANGZHOU HUAFENG BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional Brucella detection method is far from meeting the requirements of modern detection due to its shortcomings such as long detection cycle, complicated procedures, and various reagents required.
The pathogenic nucleic acid detection technology represented by polymerase chain reaction (PCR) technology has some problems in practical application, such as ordinary polymerase chain reaction (PCR) technology requires special instruments, and there are easy cross-contamination and cumbersome operation process Shortcomings
Although the fluorescent real-time quantitative polymerase chain reaction (real time PCR) technology solves the problem of cross-contamination and simplifies the operation process, it requires more complex quantitative measurement instruments, so it is not suitable for on-site rapid detection
Moreover, the cost of fluorescent probes in real-time quantitative polymerase chain reaction PCR technology is relatively high, which increases the difficulty of popularization and application.
Immunological detection technology is fast, simple and low-cost, but requires high-quality and high-stability monoclonal antibodies. Otherwise, due to insufficient accuracy, it can only be used as an auxiliary detection method at present.

Method used

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  • Kit for detecting brucella and use method thereof
  • Kit for detecting brucella and use method thereof
  • Kit for detecting brucella and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] The preparation of embodiment 1 kit

[0121] (1) Synthesize oligodeoxynucleic acid primers by DNA synthesizer according to the following sequence:

[0122] Outer primer F3 (1):

[0123] CCTTTGCTGGCTGGAACT

[0124] Outer Primer B3 (1):

[0125] GCAATACCAGCCGTGAGG

[0126] Inner primer FIP (1):

[0127] GTCCTTGGACTTCTTGGCCCAGttttTCCAGCAGGACCAGATCG

[0128] Inner primer BIP (1):

[0129] GGCCTGGAAGTCAAGCAGGGttttACGGCATAACCGGGTTCA.

[0130] (2) Purchase DNA polymerase: BstDNA polymerase is placed in the container;

[0131] (3) Prepare reaction solution and primers: the reaction solution contains 2mmol / LdNTP, 25mmol / L Tris-Cl, 12.5mmol / L KCl, 12.5mmol / L (NH 4 ) 2 SO 4 , 10mmol / L MgSO 4 , 0.125% by volume TritonX-100, 1mol / L betaine, 1.2 μmol / L each of the inner primer FIP / BIP and 0.25 μmol / L each of the outer primer F3 / B3, placed in the container;

[0132] (4) Prepare the sample pretreatment solution: the sample pretreatment solution contains 20mmol / L Tris-HCl (p...

Embodiment 2

[0152] The preparation of embodiment 2 kit

[0153] (1) Synthesize oligodeoxynucleic acid primers by DNA synthesizer according to the following sequence:

[0154] Outer primer F3 (2):

[0155] CAAGACCAGCACCGTTGG

[0156] Outer primer B3 (2):

[0157] GGTTCAGGTCGTAGCCGA

[0158] Inner primer FIP (2):

[0159] GGTCCTGCTGGAAGTTCCAGCttttAGCATCAAGCCTGACGATTG

[0160] Inner primer BIP (2):

[0161] CGGTGTTGAAGGTGATGCAGGTttttTTCAAAGCCCTGCTTGACTT

[0162] (2) Purchase DNA polymerase: BstDNA polymerase is placed in the container;

[0163] (3) Prepare reaction solution and primers: the reaction solution contains 1.6mmol / LdNTP, 20mmol / L Tris-Cl, 10mmol / L KCl, 10mmol / L (NH 4 ) 2 SO 4 , 8mmol / L MgSO 4 , 0.1 volume % TritonX-100, 0.8 mol / L betaine, 2.0 μmol / L each of the inner primer FIP / BIP and 0.2 μmol / L each of the outer primer F3 / B3, placed in the container;

[0164] (4) Prepare the sample pretreatment solution: the sample pretreatment solution contains 10mmol / L Tris-HCl (pH...

Embodiment 3

[0179] The application of embodiment 3 Brucella detection kits

[0180] 1 Materials and methods

[0181] 1.1 Materials

[0182] 1.1.1 Strains

[0183] There are 28 bacterial strains used in the present invention, which are mainly derived from the Guangzhou Center for Disease Control and Prevention, clinical isolated bacterial strains and environmental isolated bacterial strains. See Table 1 for details.

[0184] Table 1 strain name and source

[0185]

[0186] 1.1.2 Main instruments and reagents

[0187] 1.2 Identification of isolated strains

[0188] 1.2.1 Cultivation of Brucella puncture-cultured strains of Brucella were resuscitated with Brucella broth, cultured at 36±1°C for 24±2 hours.

[0189] 1.2.2 Isolation and identification of clinical isolates. Smear the bacterial solution that is slightly turbid, precipitated and does not form a bacterial film directly on blood agar, liver infusion agar or Brooke's agar plate, respectively in microaerophilic (10% Carbon d...

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Abstract

The invention provides a kit for detecting brucella. The kit comprises two pairs of primers, i.e., an inner primer FIP / BIP and an outer primer F3 / B3, which adopt an OMP25 gene of the brucella as the target gene and are designed on the basis of loop-mediated isothermal amplification technology. The kit provided by the invention has the advantages that the detecting effect is more comprehensive and the loss rate is low.

Description

[0001] This application is a divisional application with filing date: November 16, 2010, application number: 201010545462.3, title of invention: Brucella detection kit and its usage method. technical field [0002] The invention relates to a biological detection reagent, in particular to a Brucella detection kit and a use method thereof. Background technique [0003] Brucella is a Gram-negative, non-motile bacterium that can survive as an intracellular parasite in many species of livestock. The principal cause of infection in humans is Brucella malta ("Maltese fever"), abortive cloth Brucellosis, Brucella bovine, sheep, Brucella suis and Brucella canis. After a person is infected, the first symptom is fever, the body temperature can reach 38-40 degrees, and it lasts for a long time, and it is in a long-term low-grade state; , Repeated many times, so brucellosis is also called undulant fever. Therefore, it is of great significance to detect Brucella accurately and rapidly. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/01
Inventor 曹以诚陈守义杜正平宋榴艳冯雪梅邓志爱陈洵杨智聪
Owner GUANGZHOU HUAFENG BIOTECH
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