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Real-time fluorescent PCR (polymerase chain reaction) kit for detecting swine Eperythrozoon in umbilical cord blood and application thereof

A porcine Eperythrozoon and real-time fluorescence technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of non-specificity, poor repeatability, not very ideal, and low specificity, and achieve specificity and reliability Strong performance, high accuracy, high accuracy effect

Pending Publication Date: 2017-02-22
HUNAN XINNANFANG CULTURE SERVICE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the actual application process, the conventional PCR method will still show non-specificity and poor reproducibility. The homology rate of Bartonella subspecies is 79.2% to 83%, and the homology with Haematospora marginalum is between 72% and 75%. Therefore, these PCR methods based on 16SrRNA gene design are not high in specificity and are prone to false positives. Positive result; in addition, the EB used in conventional PCR detection is a harmful substance, so the conventional PCR method is not a very ideal method

Method used

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  • Real-time fluorescent PCR (polymerase chain reaction) kit for detecting swine Eperythrozoon in umbilical cord blood and application thereof
  • Real-time fluorescent PCR (polymerase chain reaction) kit for detecting swine Eperythrozoon in umbilical cord blood and application thereof
  • Real-time fluorescent PCR (polymerase chain reaction) kit for detecting swine Eperythrozoon in umbilical cord blood and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 detects the composition of the real-time fluorescent PCR kit of pig Eperythrozoon in umbilical cord blood

[0052] (1) 2× fluorescent PCR reaction solution (containing enzyme): purchased from Hunan Shengxiang Biotechnology Co., Ltd.; the reaction solution contains UNG enzyme system, which can effectively solve the phenomenon of amplification pollution in the amplification process, and has strong anti-pollution ability, etc. ;

[0053] (2) Amplification primer set oxaA-F and oxaA-R: synthesized by Shanghai Sangon Bioengineering Co., Ltd., prepared with DEPC water to a concentration of 10 μM.

[0054] Upstream amplification primer oxaA-F sequence: 5'-TTGGAAGTCAAGTGAGAACAA-3', which is the sequence of SEQ ID NO:1;

[0055] Downstream amplification primer oxaA-R: 5'-CTTCTGGATATAAATTCCTACTAGAAA-3', which is the sequence of SEQ ID NO:2;

[0056] (3) Specific fluorescent probe oxaA-P: synthesized by Huada Genomics Co., Ltd., prepared with DEPC water to a concent...

Embodiment 2

[0066] Example 2 The method of using the real-time fluorescent PCR kit for detecting porcine Eperythrozoon in umbilical cord blood

[0067] The method for using the kit of the present invention specifically includes the following steps: (1) sample collection; (2) sample processing; (3) DNA extraction; (4) real-time fluorescent RT-PCR: utilize the specific primers and probes designed by the present invention to carry out Real-time fluorescent RT-PCR detection; (5) Judgment result.

[0068] (1) Sample collection

[0069] 1. Take a clean penicillin bottle and cork, wash it, boil and sterilize it for 30 minutes, dry it and collect it for later use;

[0070] 2. When the piglets are born, squeeze the "cord blood" of all the piglets delivered by each sow into a clean penicillin bottle, 3-5 drops per piglet, and squeeze the "cord blood" into the penicillin bottle bottle, sealed;

[0071] Precautions: ①It is necessary to collect all the piglets from the same litter sow to avoid missed...

Embodiment 3

[0084] Example 3 Verification of the real-time fluorescent PCR kit for detecting porcine Eperythrozoon in umbilical cord blood

[0085] 1. Kit amplification efficiency verification

[0086] Perform a 10-fold serial dilution of the positive control clone plasmid pEASY-oxaA to make its copy number: 10 8 -10 1 copies / μl, each gradient was repeated three times for real-time fluorescent PCR, and a standard curve was made according to the amplification results.

[0087] figure 1 It is the gradient amplification standard curve (FAM channel) of 10-fold dilution of the positive control of the present invention, wherein the parameters of the standard curve are as follows: slope: -3.20, intercept: 40.57, correlation coefficient: 0.999, amplification efficiency: 1.05. It shows that the amplification efficiency of the kit for detecting porcine Eperythrozoon is close to 100%.

[0088] 2. Sensitivity test

[0089] The positive control clone plasmid pEASY-oxaA with 10-fold gradient dilut...

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Abstract

The invention discloses a real-time fluorescent PCR (polymerase chain reaction) kit for detecting swine Eperythrozoon in umbilical cord blood and application thereof; the kit is a real-time fluorescent PCR kit developed according to swine Eperythrozoon membrane protein OxaA gene for the first time and comprises Taqman fluorescent probe and primer designed for swine Eperythrozoon OxaA gene sequences; sequences of the primer are shown as in SEQ ID NO: 1 and SEQ ID NO:2, and a sequence of the fluorescent probe is shown as in SEQ ID NO: 3. The real-time fluorescent PCR kit has the advantages of high specificity, high sensitivity, high accuracy and good repeatability, and is suitable for high-throughput sample detection. The kit of the invention is suitable for detecting Eperythrozoonosis of swine umbilical cord blood, and is also suitable for detecting conventional blood and tissue samples, guiding the prevention of such disease, providing molecular epidemiological investigation and assessing therapeutic effect; in addition, through umbilical cord blood it is also possible to monitor female piglets at the same time, and the kit is important to the early quick diagnosis and preventive control of Eperythrozoonosis.

Description

technical field [0001] The invention relates to the technical field of molecular diagnosis of animal pathogens, in particular to a real-time fluorescent PCR kit for detecting Eperythrozoon porcine in umbilical cord blood and its application. Background technique [0002] Eperythrozoon (Eperythrozoon) is a zoonotic pathogen, which is a kind of non-culturable pathogenic microorganisms attached to the surface of human and animal red blood cells, or free in plasma, causing parasitic hosts to have anemia, fever, jaundice, etc. as the main clinical symptoms. Symptomatic Eperythozoonosis. [0003] The report on the study of eperythrozoonosis in my country is relatively late. In 1982, Jin Ximin first discovered and reported Eperythrozoonosis in rabbits. In the same year, Xu Yaocheng and others reported "porcine erythroderma" in southern Jiangsu, which was later confirmed to be caused by Eperythrozoon porcine infection. In 1991, Tai Xiuzhen and others in the Inner Mongolia Autonom...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q1/689C12Q2563/107C12Q2545/113
Inventor 喻正军李增强石建廖娟红
Owner HUNAN XINNANFANG CULTURE SERVICE CO LTD
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