Primer, probe, kit and method for detecting streptococcus suis based on digital PCR technology

A technology for the detection of Streptococcus suis, which is applied in the field of molecular biology to achieve the effects of precise virus content, high sensitivity, and precise absolute quantification

Inactive Publication Date: 2018-11-30
JINAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the existing technology does not use digital PCR technology to carry out relevant research on the absolute quantitative detection of Streptococcus suis, whether it is feasible, and there are no finished kits suitable for industrialization.

Method used

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  • Primer, probe, kit and method for detecting streptococcus suis based on digital PCR technology
  • Primer, probe, kit and method for detecting streptococcus suis based on digital PCR technology
  • Primer, probe, kit and method for detecting streptococcus suis based on digital PCR technology

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Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1 The establishment of the kit based on digital PCR technology absolute quantitative detection Streptococcus suis

[0047] A kit for the absolute quantitative detection of Streptococcus suis based on digital PCR technology, including primer sets, 2×ddPCRSupermix, RNase-free ultrapure water, bacterial total DNA extraction reagents, positive controls and negative controls.

[0048] (1) Design of primers for digital PCR amplification: primers were designed with the specific conserved sequence of Streptococcus suis type 1-32 as the target gene. The primer sequences are listed in Table 1.

[0049] Table 1 Primer sequence list

[0050]

[0051] (2) The molar ratio of FP primer, BP primer and probe in the primer set is 4:4:2.

[0052] (3) 2×ddPCR Supermix contains: 2×PCR Buffer, 2×Taq DNA Polymerase, 16μM FP, 16μM BP primer, 20μM probe, 400μM dNTP mix and 2×RNase Cocktail.

[0053] (4) The positive control is Streptococcus suis DNA, and the negative control is...

Embodiment 2

[0055] Embodiment 2 Absolute Quantitative Detection Method for Streptococcus suis Based on Digital PCR Technology

[0056] The method utilizing the test kit of embodiment 1 to detect Streptococcus suis comprises the steps:

[0057] (1) Extraction of bacterial DNA:

[0058] 1) Take 1 g of the sample to be tested, add 600 μL of lysate A to grind, vortex and mix for 20 seconds, and let stand at room temperature for 10 minutes;

[0059] 2) Transfer the mixture to an adsorption column and centrifuge at 12,000×g for 30-60 seconds;

[0060] 3) Discard the liquid in the collection tube, add 500 μL of washing solution B to the adsorption column, and centrifuge at 12,000×g for 30-60 seconds;

[0061] 4) Discard the liquid in the collection tube, add 500 μL washing solution C to the adsorption column, and centrifuge at 12,000×g for 30-60 seconds;

[0062] 5) Discard the liquid in the collection tube, and centrifuge at 12,000×g for 2 minutes to dry the column;

[0063] 6) Transfer the...

Embodiment 3

[0081] Embodiment 3 digital PCR kit specific experiment

[0082] Use the kit of the present invention to detect clinically obtained samples of pseudorabies virus, porcine circovirus type 2, porcine infectious pleuropneumoniae, Haemophilus parasuis, Escherichia coli, and Salmonella samples respectively, and set positive control and negative control samples , a total of 8 samples, all samples were verified by sequencing method, the test results are shown in figure 1 .

[0083] The experimental results show that after the positive control is amplified by digital PCR, positive droplets appear above the threshold line, which means amplification. However, pseudorabies virus, porcine circovirus type 2, Actinobacillus pleuropneumoniae, Haemophilus parasuis, Escherichia coli, Salmonella and negative controls are below the threshold line, that is, there is no amplification (see figure 1 ). It can be seen that the detection of Streptococcus suis by using the digital PCR kit of the pre...

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Abstract

The invention discloses a primer and probe, a kit and a method for detecting streptococcus suis based on the digital PCR technology. The primer and probe include a forward primer FP, a backward primerBP and a Probe. The detection kit includes 2 x ddPCR Supermix, ultrapure water without RNA enzymes, a bacterial total DNA extraction reagent, negative control and positive control. The detection method comprises the steps that DNA of a sample to be tested is extracted, the DNA of the sample to be tested is quantitatively detected by the microdrop digital PCR technology, a copy number obtained byamplification analysis is used for determining whether the sample to be tested contains the streptococcus suis or not, and the content of the streptococcus suis is determined. The primer and probe, the kit and the method for detecting the streptococcus suis based on the digital PCR technology have the advantages of accuracy, sensitivity, wide applicability, no dependence on a standard curve, realization of absolute quantification and the like, can realize early detection, early treatment and early prevention of the diagnosis of the streptococcus suis disease, and can effectively control the disease outbreak.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a primer, a probe, a kit and a method for detecting Streptococcus suis based on digital PCR technology. Background technique [0002] Streptococcus suis (SS) is a common opportunistic pathogen in pigs, which usually settles in the upper respiratory tract (especially the tonsils and nasal cavity), reproductive tract or digestive tract of pigs, and in severe cases, pigs will Symptoms such as meningitis, sepsis, pneumonia and arthritis occur, which can lead to sudden death in young pigs. Streptococcus suis can be divided into 35 serotypes (types 1-34 and type 1 / 2) according to the different characteristics of streptococcus capsular antigens, among which types 1, 2, 7, and 9 are pathogenic bacteria of pigs. And it can also cause human infection. Serious diseases such as meningitis, sepsis, pneumonia, arthritis, endocarditis, peritonitis and endophthalmitis will also occur af...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/46
CPCC12Q1/686C12Q1/689C12Q2563/159
Inventor 李丽丽温雯孟赫诚石磊陈洵
Owner JINAN UNIVERSITY
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