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Kit for absolutely quantitatively detecting Toxoplasma gondii based on digital PCR and detection method thereof

A technology of absolute quantification and detection method, applied in the direction of microorganism-based methods, microorganism determination/inspection, biochemical equipment and methods, etc., to achieve high sensitivity

Inactive Publication Date: 2017-02-22
吉林省畜牧兽医科学研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 1992, Sykes et al. used limited dilution of samples to obtain a single template molecule, and calculated the amplification signal after PCR in order to accurately determine the number of starting molecules. Although the concept of "digital PCR" was not clearly proposed, the digital PCR method has been established. The basic experimental process, which is the prototype of digital PCR

Method used

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  • Kit for absolutely quantitatively detecting Toxoplasma gondii based on digital PCR and detection method thereof
  • Kit for absolutely quantitatively detecting Toxoplasma gondii based on digital PCR and detection method thereof
  • Kit for absolutely quantitatively detecting Toxoplasma gondii based on digital PCR and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Design and screening of primers and probes

[0054] 1. Design of primers and probes of the present invention: according to the Toxoplasma gondii gene sequence published by Genbank (GenBank: AF179871.1) as the target gene, specific primers and probes suitable for ddPCR were designed.

[0055] This example gives the process of screening the best primers, select several pairs of alternative primers designed by software for screening, the alternative primers are as follows, see Table 1.

[0056]

[0057] 2. Screening of primers

[0058] (1) The primers are randomly matched into four pairs: F1R1, F1R2, F2R1, F2R2;

[0059] (2) Then use the dye method to do fluorescent quantitative PCR, PCR system formula: 2×SYBR Green Supermix 10μL, upstream primer, downstream primer 1μL, ddH 2 O 3 μL, positive template 5 μL. The PCR amplification program was pre-denaturation at 94°C for 10 min; denaturation at 94°C for 15 sec, annealing at 58°C for 1 min, a total of 40 cycle...

Embodiment 2

[0066] Example 2 Optimization of the annealing temperature of the PCR method for detecting Toxoplasma gondii on the ddPCR platform.

[0067] 1. Select the combination of primers and probes: F1R1+P1.

[0068] 2. Then on the ddPCR platform, ddPCR system formula: 2×ddPCR Supermix for probes 10 μL, upstream primer, downstream primer 1 μL, probe 0.5 μL, ddH 2 O 3.5 μL, positive template 4 μL, total volume 20 μL (concentration of both primers and probes is 10 μM). Do 8 replicate wells and generate microdroplets.

[0069] 3. Amplify on a PCR instrument. The PCR amplification program is 94°C for 10 minutes; denaturation at 94°C for 15 sec; . Detection on the droplet digital PCR detector.

[0070] 4. Analyzing results: Select an annealing temperature of 57-60°C according to the experimental results.

Embodiment 3

[0071] The optimization experiment of embodiment 3 primers, probe concentration

[0072] 1. The concentration of the designed primers and probes is 10 μM, and the combination is F1R1+P1. The system configuration method is shown in Table 2.

[0073]

[0074] 2. Generate microdroplets on the ddPCR platform, transfer them to 96-well plates, and seal them with aluminum film.

[0075] 3. Amplify on a PCR instrument. The PCR amplification program is 94°C for 10 minutes; denaturation at 94°C for 15 sec; annealing at 60°C for 1 minute; a total of 40 cycles; 98°C for 10 minutes to end the reaction. Detection on the droplet digital PCR detector.

[0076] 4. Analyzing results: Select F1R1+P1 primer-probe formula according to the experimental results: 1.8 μL for primers and 0.6 μL for probes.

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Abstract

The invention relates to a kit for absolutely quantitatively detecting Toxoplasma gondii based on digital PCR and a detection method thereof; the kit is composed of a microdrop digital PCR kit and a plurality of microdrop generator sheets; the detection method is characterized by comprising: (1) extracting total DNA of a test sample; (2) using the total DNA as a template to carry out PCR amplification; (3) placing the PCR production and microdrop reader to read signals, analyzing experimental data to obtain an absolute content of Toxoplasma gondii in the sample. The kit and method can provide direct quantitation in Toxoplasma gondii detection, have no need for standard curves, have the advantages, such as good operational convenience, high speed and efficiency, good specific sensitivity and low cost, etiological diagnosis on Toxoplasma gondii can be effectively carried out, detection rate is increased, the common problems of existing Toxoplasma gondii detection kits, such as low sensitivity and high misdiagnosis rate, are solved, and the kit and method are suitable for clinical investigations and large-scale disease detection and supervision, related preventive and control measures can be collected in time, and economic loss is decreased.

Description

technical field [0001] The invention relates to a kit for detecting Toxoplasma gondii in absolute quantification based on digital PCR and a detection method thereof. [0002] The invention belongs to the technical field of biological detection and is suitable for the application in the early diagnosis and epidemic prevention of animal toxoplasmosis. Background technique [0003] Toxoplasmosis (Toxoplasmosis) is caused by Toxoplasma gondii ( Toxoplasma gondii ) caused by a zoonotic parasitic disease. The disease has a global distribution. Dogs, cats, pigs, cattle, sheep, rabbits and other domestic animals are very common to be infected with Toxoplasma gondii, and the infection rate is as high as 10% to 47.3%. The human Toxoplasma gondii infection rate is generally 20% to 50%, as high as 94%. About 1 / 3 of the world's people are infected with Toxoplasma gondii. The infection rate of Toxoplasma gondii in the Chinese population is 5% to 20%. Toxoplasma gondii can cause seriou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/90
CPCC12Q1/6851C12Q2531/113C12Q2563/159
Inventor 曹利利宫鹏涛姚新华郭衍冰董航苑淑贤
Owner 吉林省畜牧兽医科学研究院
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