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Kit and method for identifying types of porcine derived eperythrozoons and application of kit and method

A technology of Eperythrozoon and small Eperythrozoon, which is applied in the field of kits for identifying types of Eperythrozoon from porcine

Inactive Publication Date: 2015-11-04
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

PCR-RFLP technology has been widely used in the identification of microbial species, but so far, there is no report on the identification of Eperythrozoon species by PCR-RFLP technology at home and abroad

Method used

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  • Kit and method for identifying types of porcine derived eperythrozoons and application of kit and method
  • Kit and method for identifying types of porcine derived eperythrozoons and application of kit and method
  • Kit and method for identifying types of porcine derived eperythrozoons and application of kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 PCR Amplification of the Pig-derived Eperythrosome 16S rRNA Gene

[0036] 1. Primer Synthesis

[0037] According to the report of Messick et al. (1999), the universal primer pair fHf1 / rHf2 can amplify the nearly full-length 16S rRNA gene of hemotrophic bacteria such as Eperythrozoon or Bartonella hemotrophs and most eubacteria. Accordingly, the upstream primer F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 3) was designed, located at 1bp-20bp of the nucleotide sequence of M. suis (accession number NR_103930.1), M.parvum (accession number CP006771. 1) 11bp-30bp of the 16S rRNA sequence; downstream primer R: 5'-TACCTTGTTACGACTTAACT-3' (SEQ ID NO: 4), located at 1450bp-1469bp of the nucleotide sequence of M. suis (accession number NR_103930.1) , the 1460bp-1479bp of the 16S rRNA sequence in M. parvum (accession number CP006771.1). Wherein, the 1469bp16S rRNA sequence amplified by Mycoplasma suis (GenBank Accession No. NR_103930.1) is shown in SEQ ID NO: 1, and the a...

Embodiment 2

[0054] Example 2 Analysis of 16S rRNA gene cleavage sites in pig-derived Eperythrozoon and the establishment of RFLP technology

[0055] 1. Analysis of the cleavage sites of the 16S rRNA gene of porcine Eperythrosome

[0056] A total of 16 known 16S rRNA nucleotide sequences of different species of Eperythrozoon were downloaded from GenBank, together with the nucleotide sequence and small A total of 18 nucleotide sequences of the Eperythrozoon JD-71 isolate were loaded into DNAMAN to analyze all restriction sites. The selected eperythrozoons and their accession numbers are: porcine eperythrozoon (accession numbers AB610847, ESU88565, FN984917), small eperythrozoon (accession numbers AB610845, AB610846, AB610850, JX489599), cat eperythrozoon (M. haemofelis, accession number AF178677), canine Eperythrozoon (M.haemocanis, accession number AF197337), Wen's Eperythrozoon (M.wenyonii, accession number AY946266), sheep Eperythrozoon (M.ovis, accession number AF338268), Eperythrozoo...

Embodiment 3

[0064] Example 3 Identification of the composition of the pig-derived Eperythrozoon species kit and the application of the kit to the detection of field samples

[0065] 1. The composition of the kit

[0066] Assemble the primer pair, Sca I or Ssp I restriction endonuclease, restriction endonuclease buffer, PCR reaction buffer and polymerase into a kit, and store at -20°C after assembly.

[0067] 2, the application of kit of the present invention

[0068] Apply the kit of the present invention, and use the established PCR-RFLP technique to detect 40 field positive DNA samples of Eperythrozoon from pigs stored in the laboratory.

[0069] Results: Sca Ⅰ and Ssp Ⅰ digestion of 16S rRNA gene PCR products of field samples successfully obtained fragments of different sizes, and the digestion results of PCR products of some samples were as follows: Figure 4 and Figure 5 . Figure 4 It is the result of Sca Ⅰ enzyme digestion of the 16S rRNA gene PCR product of porcine Eperythroz...

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Abstract

The invention discloses a kit for indentifying the types of porcine derived eperythrozoons. The kit comprises a primer pair for amplifying a porcine derived eperythrozoon 16S rRNA gene sequence shown by SEQ ID NO:1 or SEQ ID NO:2 and restriction endonuclease Sca I or Ssp I. The kit for identifying the types of the porcine derived eperythrozoons on the basis of the 16S rRNA gene can be used for correctly and quickly identifying two types of eperythrozoons infecting pigs, including a mycoplasma suis and an M.parvum, is beneficial for the works of clinical diagnosis of porcine derived eperythrozoonosis, molecular epidemiology survey and pig farm purification, and has an important significance for early rapid diagnosis, prevention, control and purification of the porcine derived eperythrozoonosis.

Description

technical field [0001] The invention belongs to the technical field of detecting mycoplasma infection in animal blood, and in particular relates to a kit, a method and an application thereof for identifying types of Eperythrozoon from pigs. Background technique [0002] Eperythrozoonosis is a blood infectious disease characterized by hemolytic anemia and jaundice caused by Mycoplasma spp. parasitizing on the surface of red blood cells, plasma and bone marrow of various animals and humans. Eperythrozoon is an important zoonotic pathogen, which is widely distributed in animals. Pigs, cattle, sheep, dogs, rabbits, chickens and humans are all susceptible animals to Eperythrozoon. The rate is higher. Eperythrozoonosis of pigs not only reduces the yield and quality of livestock products, but also reduces the fertility of pigs. Clinically, it is often co-infected with other diseases, which makes diagnosis difficult, leading to severe clinical symptoms and even death of animals, hi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/35
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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