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Method for detection and differential diagnosis of Brucella in aerosol

A technology of Brucella and differential diagnosis, applied in the field of detection and differential diagnosis of Brucella in aerosols of farms

Active Publication Date: 2014-08-13
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no application of SYBRGreenⅠreal-time fluorescent quantitative PCR technology to carry out research reports on Brucella in farm aerosol

Method used

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  • Method for detection and differential diagnosis of Brucella in aerosol
  • Method for detection and differential diagnosis of Brucella in aerosol
  • Method for detection and differential diagnosis of Brucella in aerosol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The design and synthesis of embodiment 1 primer

[0055] The comparison of the VirB10 gene sequence of bovine (GenBank: AF226278.1, sequence 10 in the sequence listing), sheep (GenBank: DQ861303.1) and pig (GenBank: AF141604.1) found that it is highly conserved, see figure 1 . According to the conserved sequence of Brucella bovis VirB10 gene in GeneBank, design full-length amplification primer (as shown in SEQIDNO.1,2), be used for the construction of positive standard plasmid (sequence 1,2 in table 1); Adopt PrimerExpress3 .0 software, design a pair of specific primers (as shown in SEQIDNO.3,4), be used for Brucella universal detection (sequence 3,4 in table 1). According to the Brucella bovis AlkB gene and the IS711 sequence (GenBank: AF148682.1, shown in SEQ ID NO.11) respectively in GeneBank, the Brucella caprica insertion sequence IS711 (GenBank: JF939171.1, shown in SEQ ID NO.12 Shown), the chromosomal sequence of Brucella suis S1330 strain (GenBank: 1618935~161...

Embodiment 2

[0058] The establishment of Brucella passing type detection method in embodiment 2 aerosol

[0059] (1) Establishment of SYBRGreenⅠreal-time fluorescent quantitative PCR method

[0060] 1. Preparation of samples to be tested

[0061] Genomic DNA was extracted according to the instructions of the bacterial DNA extraction kit.

[0062] 2. Preparation of standard products

[0063] Carry out PCR amplification with the DNA of the Brucella bovis live vaccine (A19 strain) that step 1 obtains as template, and reaction system is 50 μ L: 10 * LAPCRBufferII (Mg 2+ Plus) 5 μL, 2.5mMdNTPMixture8 μL, LATaq0.5 μL (5U / μL), template 2 μL, 10 μmol / L of VirB10 gene full-length upstream and downstream primers VirB10-1 (sequence 1 and sequence 2 in Table 1) each 2 μL, sterilized supernatant Add pure water to 50 μL. The reaction program was pre-denaturation at 94°C for 3min, followed by 30 cycles at 94°C for 30s, 55°C for 30s, and 72°C for 1min; extension at 72°C for 10min, and finally stopped ...

Embodiment 3

[0085] Establishment of method for differential diagnosis of bovine species, sheep species and swine brucella in embodiment 3 aerosol

[0086] Respectively with the DNA of the Brucella bovis live vaccine A19 strain, the Brucella sheep species live vaccine M5-90 strain and the Brucella suis live vaccine S2 strain as a template, simultaneously use Abortus primers (such as SEQ ID NO.5 , shown in 6), Melitensis primer (as shown in SEQIDNO.6,7) and Suis primer (as shown in SEQIDNO.8,9), carry out primer concentration, annealing according to 3 and 4 of step (1) in embodiment 2 The optimization of temperature and reaction condition, other methods are with embodiment 2. application The TmCalling analysis method of 480II GeneScanningSoftwareVersion1.5 calculates the Tm values ​​of Brucella bovine, sheep and porcine species respectively, and makes differential diagnosis through the difference of Tm values. The results are as follows Figure 8 , 9 , 10, one specific melting curve wa...

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Abstract

The invention discloses a method for detection and differential diagnosis of Brucella in an aerosol. The method comprises the following steps: (1) acquisition of an aerosol sample; (2) extraction of genome total DNA of the aerosol sample; (3) detection: a step of carrying out PCR amplification by using specific primers and a kit; (4) establishment of a standard curve and a melting curve: a step of establishing the standard curve of pEASY-T3-VirB10 positive standard plasmid and the melting curve of an amplification system; and (5) judgment: a step of judging whether the aerosol sample contains Brucella. The invention further discloses the specific primers (as shown in SEQ ID No. 3 to 9) and the kit (composed of the specific primers, the pEASY-T3-VirB10 positive standard plasmid, an SYBRGreenI real-time fluorescent quantitative PCR reagent and ddH2O). The method provided by the invention is applicable to detection and identification of Brucella in the aerosol in a culturing farm and to detection and identification of samples like clinical blood, serum, milk and tissue.

Description

technical field [0001] The invention relates to a method for detecting and differentially diagnosing Brucella in the aerosol of farms, belonging to the field of biological detection. Background technique [0002] Aerosol refers to a multiphase system composed of solid and / or liquid particles suspended in gas and gas carrier. According to the composition of particles, it can be divided into biological aerosol and chemical aerosol. In the aerosol system, living aerosol particles and active particles and various plasmids released into the air by living organisms are collectively referred to as bioaerosols, also known as microbial aerosols, which are mainly divided into bacteria Aerosols, virus aerosols and fungal aerosols are closely related to human and animal health. The distribution of microbial aerosols in the farm environment can cause the outbreak and prevalence of infectious diseases transmitted by the respiratory tract, and bring serious harm to animal husbandry produ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6851C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 何洪彬赵贵民王洪梅
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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