Pig-sourced eperythrozoon fluorogenic quantitative PCR detection kit and application thereof

A porcine eperythrozoon and eperythrozoon technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve problems such as the need for further improvement of specificity and sensitivity, and achieve high sensitivity, strong specificity, good repeatability

Active Publication Date: 2015-10-14
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its specificity and sensiti

Method used

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  • Pig-sourced eperythrozoon fluorogenic quantitative PCR detection kit and application thereof
  • Pig-sourced eperythrozoon fluorogenic quantitative PCR detection kit and application thereof
  • Pig-sourced eperythrozoon fluorogenic quantitative PCR detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Amplification and sequence analysis of the porcine Eperythrozoon a1 gene

[0035] 1. Porcine Eperythrozoon infection positive blood

[0036] Pig fresh blood was collected from a slaughterhouse in Changning, Jiading, and Songjiang districts of Shanghai, and the anticoagulant trisodium citrate (1.32%) was added to the sampling tube in advance, and the ratio of blood to anticoagulant was 1:10. The blood was brought back to the laboratory, and those who were positive for porcine Eperythrozoon were tested by TaqMan Real-time PCR technology based on the g1 gene.

[0037] 2. Extraction of Genomic DNA from Pig Blood

[0038] 2.1 Blood processing

[0039] Centrifuge 41 anticoagulated blood samples positive for Eperythrozoon porcine by the TaqMan real-time PCR technique based on the g1 gene at 3000r / min for 10min, pour off the upper solution, and carefully draw the middle red blood cells (about 500μL) into the Immediately extract blood genomic DNA from sterilized 2mL...

Embodiment 2

[0063] Example 2 Establishment of Porcine Eperythrosome TaqMan Real-time Fluorescent Quantitative PCR Detection Method

[0064] 1. Design of primers and probes

[0065]The obtained and correctly identified 41 Eperythrozoon porcine a1 gene sequences were compared with the a1 gene sequence published by NCBI (accession number AM265536) using DNAMAN software (version number 7.0.2.176) for multiple sequence comparison analysis, and selected a few relative For conserved fragments, a pair of specific primers and TaqMan probes are designed and synthesized to ensure that the designed primers and probes are highly specific for comparison with other species of pathogen genes and pig genes on NCBI. The primer sequences are as follows: F: 5'-GCTGGAAAGATTGCTGGACTAGA-3' (SEQ ID NO: 4), located at the 360-382th position of the 1657bp a1 gene nucleotide sequence, R: 5'-CCTCCCCCTAGGTCAT AAACAAGTA-3' (SEQ ID NO : 5), located at the 460-484th position of the 1657bp sequence; the probe sequence i...

Embodiment 3

[0080] Example 3 Sensitivity experiment and determination of Ct cut-off threshold

[0081] The recombinant plasmid pMD18-T-a1 (sample No. 126-5) was used as a standard, and its gene copy number concentration was known to be 3.48×10 10 copies / μL. Dilute it 10 times with dilution buffer, take 3.48×10 8 copies / μL, 3.48×10 7 copies / μL, 3.48×10 6 copies / μL, 3.48×10 5 copies / μL, 3.48×10 4 copies / μL, 3.48×10 3 copies / μL, 3.48×10 2 The 8 dilutions of copies / μL and 3.48×10copies / μL were used as templates for real-time fluorescence quantitative PCR reaction, and the amplification curves and Ct values ​​corresponding to the standards of each concentration gradient were obtained. at 10 -8 dilution (i.e. 3.48 x 10 2 copies / μL concentration), followed by 2-fold dilution, a total of 6 dilutions, respectively 2 -1 ,2 -2 ,2 -3 ,2 -4 ,2 -5 ,2 -6 , to determine the negative and positive cut-off values ​​of the samples.

[0082] Results: The recombinant plasmid pMD18-T-a1 standard...

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Abstract

The invention discloses a pig-sourced eperythrozoon fluorogenic quantitative PCR detection kit which includes: a TaqMan probe which is designed on the basis of an a1 gene sequence, represented as the Seq ID No.1, of the pig eperythrozoon; and a primer pair which is designed on the basis of the gene sequence, represented as the Seq ID No.1, of the pig eperythrozoon. The pig-sourced eperythrozoon fluorogenic quantitative PCR detection kit based on a1 gene is strong in specificity, high in sensitivity and good in repeatability, can quickly and accurately detect whether the eperythrozoon exists in a pig blood sample at a high throughput, can be used in clinical diagnosis, molecular epidemiological investigation and therapeutic effect evaluation of pig eperythrozoon diseases and pig farm cleaning, and has significant meaning on early-stage quick diagnosis, prevention and control and purification of pig eperythrozoon diseases.

Description

technical field [0001] The invention belongs to the technical field of detecting mycoplasma infection in animal blood, and in particular relates to a porcine-derived Eperythrozoon fluorescent quantitative PCR detection kit and application thereof. Background technique [0002] Eperythrozoon porcine is a member of the Mycoplasma hemophilus family that cannot be cultured in vitro, and generally parasitizes on the surface of porcine red blood cells, plasma and bone marrow. Eperythrozoon infection of porcine origin is widely distributed all over the world, causing severe economic losses to the swine industry. Its acute infection can lead to severe bacteremia, acute red blood cell hemolysis, and sometimes death of young piglets, abortion of pregnant sows, etc. For chronically infected pigs, the blood bacteria content is low, and the clinical symptoms are variable, such as mild jaundice, sub-health, slow growth rate, low production capacity or susceptibility to other infectious d...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06
Inventor 陈兆国王向佩周鹏米荣升黄燕
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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