Multiplex PCR primer probes and kit for detecting pet-derived zoonotic pathogens

A kit and pet technology, applied in the field of molecular biology, can solve the problems of increasing clinical testing workload and testing cost, reducing testing efficiency, etc., and achieve the effects of simplifying testing workload, excellent testing sensitivity, and shortening testing time

Active Publication Date: 2019-04-12
西安博睿康宁生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Greatly increased clinical testing workloa...

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  • Multiplex PCR primer probes and kit for detecting pet-derived zoonotic pathogens
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  • Multiplex PCR primer probes and kit for detecting pet-derived zoonotic pathogens

Examples

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Embodiment 1

[0039] Embodiment 1 is designed for the primer probe of target sequence

[0040] Amplifying Bartonella henselae (BH), Toxoplasma gondii (TG), and the target sequence of Brucella (BRU) in the embodiment, the inventor designed 3 sets of detection systems A and B respectively for each pathogen , Group C (Table 1 below), and compared and optimized the three systems.

[0041] Table 1

[0042]

[0043] The comparison test of different primer probes designed in Table 1 for 3 pathogens found that the following primers and probe combinations have stable fluorescence signal values ​​and good amplification effects, see Figure 1-Figure 3 Therefore, the present invention determines that the combination of primers and probes is the best combination for detecting Bartonella henselae (BH), Toxoplasma gondii (TG) and Brucella (BRU) by fluorescent quantitative PCR.

[0044] BH detection group A BH-ITS-F CAGCGTCCATTTGGTTGATATAAA (SEQ ID NO.1) in Table 1

[0045] BH-ITS-R GAACCGATAGTTTCATAT...

Embodiment 2

[0051] Example 2 Optimization and method establishment of multiple fluorescent PCR detection experiment parameters

[0052] (1) System annealing temperature optimization: the system annealing temperature was changed from 55°C to 65°C, and the results showed that the system with an annealing temperature of 56-59°C had the best amplification effect (see Figure 4 ).

[0053] (2) Optimization of the concentration of system primers and probes: the system primers (25 μM) and probes (25 μM) were sequentially increased from 0.1 μl to 0.5 μl, each increment was 0.1 μl, and three parallel samples were made for each concentration gradient. Results The upstream and downstream primers and probes of the BH-ITS system were 0.4, 0.2, and 0.1 μl, the upstream and downstream primers and probes of the TG-529 system were 0.4, 0.2, and 0.1 μl, and the upstream and downstream primers and probes of the BRU SPP-bcsp31 system were 0.4 , 0.2, 0.1 μl, the amplification effect of the system is the best...

Embodiment 3

[0067] The specificity evaluation of embodiment 3 multiplex fluorescent PCR detection system

[0068] The multiplex fluorescent PCR method optimized in Example 2 of the present invention was used to detect 19 common pathogens and chromosomes of dogs and cats (Table 2), and the nucleic acids of BH, TG, and BRU positive clinical isolates were used as positive controls. As a result, except for the positive control, all the other pathogenic templates were negative ( Figure 5 ).

[0069] Table 2 Common pet pathogen templates

[0070]

[0071]

[0072] ICDC, Institute of Infectious Diseases Prevention and Control, Chinese Center for Disease Control and Prevention.

[0073] BMU, Peking University First Hospital Fungi Collection.

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Abstract

The present invention provides multiplex PCR primer probes and a kit for detecting pet-derived zoonotic pathogens. Bartonella henselae, toxoplasma gondii and brucella belonging to pet-derived zoonoticpathogens are subjected to gene sequence analysis and comparison, triple fluorescent PCR detection primers and probes suitable for single reaction and simultaneous detection of the three pathogens are designed, and nucleotide sequences of the primers and probes are shown in SEQ ID NO.1-9, respectively. The present invention also discloses a method and a detection kit for detecting the above 3 pathogens. The detection method simplifies detection workload of 2/3 or more of the original, and shortens detection time by about 1 hour, and detection sensitivities of the bartonella henselae, toxoplasma gondii and brucella are 5, 5 and 50 copies, respectively. The multiplex PCR primer probes have no non-specific amplification of 23 common pathogens and canine and cat chromosomes, and show good specificity.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to fluorescent quantitative PCR primers, probes and kits for detecting three pet-derived zoonoses of Bartonella henselae, Toxoplasma gondii and Brucella . Background technique [0002] With the improvement of people's living standards, the number of pets in cities has soared. Pets have gradually become an important part of many families, and even treat each other as family members, and have very close contact with humans. However, while pets bring fun to people's lives, they also bring hidden dangers that are difficult to detect. threaten human health. The control of pet diseases has become one of the common concerns of the international community. Cats and dogs are currently the most common pets in China. According to incomplete statistics, there are more than 70 kinds of pets that are directly or indirectly related to pet dogs and cats. These zoonotic pathogens are directly o...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6893C12Q1/6851C12Q1/04C12N15/11
CPCC12Q1/6851C12Q1/689C12Q1/6893C12Q2600/166C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107C12Q2545/113
Inventor 李睿蔡虎高博周梦诗
Owner 西安博睿康宁生物科技有限公司
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