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Absolute fluorescent quantitative polymerase chain reaction (PCR) primer pair, probe and method for determining growth titer of mycoplasma hyopneumoniae

A technique for mycoplasma hyopneumoniae and fluorescence quantification, which is applied in the field of molecular biology and can solve the problems of time-consuming, fast detection and poor accuracy.

Active Publication Date: 2014-02-12
WENS FOOD GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The existing M. hyopneumoniae titer assay method has the following disadvantages: (1) Color change unit (CCU) assay: the detection effect of this method is good, but due to the slow growth of M. hyopneumoniae, the CCU assay takes too long (about 10 days). left and right), unable to meet the actual needs
(2) Doubling ratio dilution ordinary PCR method: this method has fast detection speed, but poor accuracy

Method used

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  • Absolute fluorescent quantitative polymerase chain reaction (PCR) primer pair, probe and method for determining growth titer of mycoplasma hyopneumoniae
  • Absolute fluorescent quantitative polymerase chain reaction (PCR) primer pair, probe and method for determining growth titer of mycoplasma hyopneumoniae
  • Absolute fluorescent quantitative polymerase chain reaction (PCR) primer pair, probe and method for determining growth titer of mycoplasma hyopneumoniae

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Experimental program
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Effect test

Embodiment 1

[0028] Embodiment 1 Absolute fluorescence quantitative PCR measures the growth titer of Mycoplasma hyopneumoniae

[0029] The steps of the following method describe in detail the preparation of the plasmid, the determination of the concentration of the plasmid, the establishment of a standard curve for the amplification of the plasmid, and the determination of the fermentation liquid sample of Mycoplasma hyopneumoniae to be tested. Taking the determination of the titer of the Mycoplasma hyopneumoniae fermentation broth after one fermentation as an example, the absolute fluorescence quantitative PCR method of the present invention is used to detect its copy number, that is, the growth titer of the Mycoplasma hyopneumoniae fermentation broth.

[0030] 1) The Primer express software was used to design common PCR primer pairs at the 16S rRNA conserved sequence of Mycoplasma hyopneumoniae (GB︱AE017243.1︱), and the common PCR primer pairs were synthesized by Invitrogen. The common P...

Embodiment 2

[0046] Embodiment 2 Absolute fluorescence quantitative PCR repeatability experiment

[0047] Using the absolute fluorescence quantitative PCR method in Example 1, standard plasmids with different concentration gradients were used as templates, and the standard plasmids with different concentration gradients were measured 5 times in the same reaction, and the coefficient of variation (CV) of each repeated sample was calculated. The experimental results showed (Table 2) that the coefficient of variation of the Ct value of each dilution gradient repeated sample was less than 2%, showing that the absolute fluorescence quantitative PCR of the present invention has good reproducibility.

[0048] Table 2 Fluorescent quantitative PCR repeatability experiment

[0049] copy

[0050] ×10 -5

Embodiment 3

[0051] Embodiment 3 Absolute fluorescence quantitative PCR primer pair specific experiment

[0052] Use the bacterial genomic DNA extraction kit to extract the genomic DNA of Mycoplasma hyopneumoniae as a positive control, and simultaneously extract the DNA of Mycoplasma hyorhina, Escherichia coli, Salmonella, Bacillus subtilis, and Staphylococcus aureus for specific detection, using the absolute fluorescent quantitative PCR in Embodiment 1 Primer pair and probe and method, the result all does not have amplification curve except positive control, illustrates that the absolute fluorescence quantitative PCR primer pair of the present invention has good specificity, see image 3 .

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Abstract

The invention discloses an absolute fluorescent quantitative polymerase chain reaction (PCR) primer pair, an absolute fluorescent quantitative PCR probe and an absolute fluorescent quantitative PCR method for determining the growth titer of mycoplasma hyopneumoniae. The sequences of the absolute fluorescent quantitative PCR primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2. The sequence of the absolute fluorescent quantitative PCR probe is shown as SEQ ID NO.3. The absolute fluorescent quantitative PCR method for determining the growth titer of the mycoplasma hyopneumoniae comprises the following steps: (1) extracting the deoxyribose nucleic acid (DNA) of the mycoplasma hyopneumoniae; (2) performing common PCR amplification to obtain a target fragment, and designing the absolute fluorescent quantitative PCR primer pair and the absolute fluorescent quantitative PCR probe in the middle of the target fragment; (3) preparing plasmids by connecting the target fragment and a PMD-18-T carrier, transfecting competent cells, and extracting plasmids of a positive bacteria solution; (4) calculating an initial plasmid copy number by using the plasmids extracted in the step (3) as standard substances; and (5) performing fluorescent quantitative PCR. According to the absolute fluorescent quantitative PCR method for determining the growth titer of the mycoplasma hyopneumoniae, the copy number of target genes, namely the growth titer of the mycoplasma hyopneumoniae, can be quickly and accurately determined.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to an absolute fluorescent quantitative PCR primer pair, a probe and a method for measuring the growth titer of mycoplasma hyopneumoniae. Background technique [0002] Mycoplasma pneumonia of swine (MPS), also known as swine endemic pneumonia (Swine enzootic pneumonia), commonly known as "swine panting disease". The disease exists in all parts of the world, with high infection rate and low death rate. One of the most important diseases of pig industry economic loss (Li Yanming, Zhang Ying. Biological research progress of Mycoplasma hyopneumoniae [J]. Advances in Veterinary Medicine, 2003,24(3):25-27). According to preliminary statistics, the direct economic loss caused by the disease to my country's pig industry is as high as 4 billion yuan every year (Shen Qingchun, Ning Yibao, Qin Qingsong. Research progress of Mycoplasma hyopneumoniae [J]. Chinese Journal of Veterinary Medicine,...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/35
CPCC12Q1/6851C12Q1/686C12Q2545/114C12Q2531/113
Inventor 卢围周庆丰宋延华王东东孔意端宋志军廖承球李薇
Owner WENS FOOD GRP CO LTD
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