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Primer and kit for detecting eperythrozoon suis, small eperythrozoon and undefined species eperythrozoon

A technique for porcine eperythrozoon and eperythropoiesis, which is applied in the determination/inspection of microorganisms, microorganisms, and microorganism-based methods, etc., can solve the problems of hysteresis, false positives, research difficulties, etc. The effect of saving reagent consumables and improving detection efficiency

Active Publication Date: 2014-08-27
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the haemophilus mycoplasma cannot be purely cultured, it has caused great difficulties in its research
At present, clinical diagnosis mainly relies on microscopic examination of fresh blood pressure film and Giemsa stained film. These methods have hysteresis and significant false positives. Laboratory diagnostic methods include enzyme-linked immunosorbent assay (ELISA) for detection of antibodies and detection of pathogens. Polymerase chain reaction (PCR) diagnostic method, but the pathogen that existing diagnostic method is mainly aimed at is porcine Eperythrozoon MS, and the diagnostic method of other two pathogens MP and CMH has not yet been reported

Method used

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  • Primer and kit for detecting eperythrozoon suis, small eperythrozoon and undefined species eperythrozoon
  • Primer and kit for detecting eperythrozoon suis, small eperythrozoon and undefined species eperythrozoon
  • Primer and kit for detecting eperythrozoon suis, small eperythrozoon and undefined species eperythrozoon

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Experimental program
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Effect test

Embodiment 1

[0063] The composition of embodiment 1 kit

[0064] 1. Composition of the kit

[0065] 1) DNA extraction reagent; Whole blood DNA extraction kit (Blood DNA Mini kit, Cat.No.3001050) of Hangzhou Xinjing Biotechnology Development Co., Ltd.;

[0066] 2) DNA standard product: T vector plasmid containing porcine Eperythrozoon target gene and T vector plasmid containing unspecified Eperythrozoon target gene;

[0067] 3) PCR reaction solution, which includes: PCR buffer, dNTP, Taq DNA polymerase, sense primers and antisense primers for detecting porcine Eperythrozoa and small Eperythrozoa, and sense primers and antisense for detecting unspecified Eperythrozoa Primer;

[0068] Preferably used: 1×PCR buffer, sense primer 0.2 μM, antisense primer 0.2 μM for detection of porcine Eperythrozoon and small Eperythrozoon and unspecified Eperythrozoon sense primer 0.2 μM, antisense primer 0.2 μM, Taq DNA polymerization Enzyme 1U, dNTP0.1mM each;

[0069] The Taq DNA polymerase is TakaRa Ta...

Embodiment 2

[0080] The specificity test of embodiment 2 kit

[0081] 1. Materials, see Table 2:

[0082]

[0083]

[0084] 2. Method:

[0085] Utilize the kit usage method described in the present invention to detect common part related bacterial diseases and parasitic diseases of pigs, simultaneously with 3 * 10 6 The positive plasmid at the copy / μL concentration is a positive control, and its specificity is verified.

[0086] 3. Results

[0087] The kit of the present invention detects five kinds of porcine bacterial diseases and parasitic diseases listed in Table 2, and only the positive control has amplified bands. The results show that the kit of the present invention has good specificity.

Embodiment 3

[0088] Sensitivity detection of embodiment 3 kit

[0089] In order to verify the detection sensitivity of the kit of the present invention, with 10-fold diluted DNA standard substance (3 × 10 6 ~3×10 1 copy / μL) as the template, add 5 μL to each dilution of the template, and set up a negative control at the same time, according to the established method, carry out the amplification reaction on the PCR machine, when the template is 3 × 10 2 When copy / μL, the electrophoresis results showed two specific bands of 532bp and 384bp, when the template was 3×10 copies / μL, the electrophoresis results showed a specific band of 384bp, indicating that the method established in this experiment can detect the lowest to CMH for 3 x 10 2 copies / μL of DNA, MS and MP were detected as 3×10 copies / μL of DNA.

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Abstract

The invention discloses a primer and a kit for detecting eperythrozoon suis, small eperythrozoon and undefined species eperythrozoon. The primer for detection is in SEQ ID NO:1 to SEQ ID NO:4 in a sequence tablet. The invention also provides a kit for detecting eperythrozoon suis, small eperythrozoon and undefined species eperythrozoon. The selected primer is strong in specificity, and can simultaneously detect three pathogens, so that the cost is saved, the steps are reduced, and the efficiency is improved. The method disclosed by the invention has the characteristics of high specificity, high sensitivity, high efficiency and good stability.

Description

technical field [0001] The invention relates to a primer and a detection kit for detecting Eperythrozoon porcine, Eperythrozoon small and unspecified species. Background technique [0002] Eperythrozoonosis in pigs mainly causes symptoms such as fever, anemia, paleness, jaundice, and sow abortion in piglets. It is often mixed with other pig diseases and causes serious harm, and it is very difficult to prevent and control. In recent years, studies have shown that there are two types of Eperythrozoon that infect pigs, Mycoplasma suis MS and Mycoplasma parvum MP, which originally belonged to the Plastidaceae of the order Rickettsia. According to the sequence analysis of 16Sr RNA gene and the observation result of electron microscope, Eperythrozoa was divided into Mycoplasma haemophilus of Mycoplasma genus. We isolated a third neoeperythrozoon pathogen that infected pigs from Eperythrozoon-infected pigs, and published its 16S rRNA and rnpB gene sequences on Genbank (accession n...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/35
CPCC12Q1/686C12Q2537/143
Inventor 付媛石团员方维焕徐丽华袁秀芳李军星卢福庄张雪娟舒琦艳魏巍周永学
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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