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Fluorescent quantitation PCR (photo-conductive relay) primer combination and method for rapid detection of dog babesia and kit with fluorescent quantitation PCR primer combination

A canis Babesia, fluorescence quantitative technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms and other directions, can solve the problems of detection speed and detection sensitivity need to be improved, and achieve simple method, strong specificity, good reproducibility

Active Publication Date: 2017-06-27
洛阳禹迹生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection speed and detection sensitivity of this method need to be improved.

Method used

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  • Fluorescent quantitation PCR (photo-conductive relay) primer combination and method for rapid detection of dog babesia and kit with fluorescent quantitation PCR primer combination
  • Fluorescent quantitation PCR (photo-conductive relay) primer combination and method for rapid detection of dog babesia and kit with fluorescent quantitation PCR primer combination
  • Fluorescent quantitation PCR (photo-conductive relay) primer combination and method for rapid detection of dog babesia and kit with fluorescent quantitation PCR primer combination

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Effect test

Embodiment 1

[0040]The fluorescent quantitative PCR primer combination (as shown in SEQ ID NO:1,2) that is used for rapid detection of Babesia canis in the present embodiment, the nucleotide sequence is as follows:

[0041] Forward primer: 5'-TGTTGCAGTTAAAAAGCTCGTAGTTG-3',

[0042] Reverse primer: 5'-TCCATGCTGAAGTATTCAAGACACA-3'.

Embodiment 2

[0044] The kit used for rapid detection of Babesia canis in this example includes: 2×SYBR Master Mix 200 μL (purchased from Roche), 10 μL each of 20 μmol / L forward and reverse primers, 500 μL of deionized water, and Bessie worm 18S rRNA standard, DNAMarker and instruction manual;

[0045] The nucleotide sequence of the fluorescent quantitative PCR primer combination is as follows:

[0046] Forward primer: 5'-TGTTGCAGTTAAAAAGCTCGTAGTTG-3',

[0047] Reverse primer: 5'-TCCATGCTGAAGTATTCAAGACACA-3'.

[0048] Instructions for use: Use the total DNA of the blood sample to be tested as a template, and use the combination of fluorescent quantitative PCR primers to perform fluorescent quantitative PCR amplification; use the accumulation of fluorescent signals to monitor the entire PCR process in real time, and judge whether the sample contains canine by whether there is a specific amplification curve. Babesia; the reaction system is 20μL system: 2×SYBR Master Mix 10.0μL, 20μmol / L for...

Embodiment 3

[0076] 1. Incidence

[0077] A Teddy dog ​​in Henan, female, 4 years old, was immunized according to the normal procedure. It is understood that the dog had been active in the mountains one week before the onset of the disease. When he returned home to bathe the dog, he found many ticks. The owner removed the ticks with his hands without knowing it. When he was seriously ill and was admitted to the hospital for treatment One tick was still found on the medial femur. According to the clinical observation, the dog was depressed, eating little or not, the urine was yellowish brown, the body temperature was as high as 40°C, the visible mucous membrane was pale, highly anemic, and the urine was the color of strong tea. No Babesia was found in the blood under the microscope. It is recommended to perform fluorescent quantitative PCR detection. The operation process is as follows:

[0078] (1) Sample DNA extraction

[0079] First, 300 μL of anticoagulated blood was collected from t...

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Abstract

The invention discloses a fluorescent quantitation PCR (photo-conductive relay) primer combination and method for rapid detection of dog babesia and a kit with the fluorescent quantitation PCR primer combination and belong to the technical field of biological detection. The fluorescent quantitation PCR primer combination is designed for a dog babesia 18S RNA sequence, and a primer is high in specificity, free of interference of other nonobjective genes and capable of determining the transcriptional level of the dog babesia and quantifying the number of the dog babesia rapidly efficiently and accurately. The dog babesia is quantitively detected with the fluorescent quantitation PCR method which is simple, accurate and free of pipe contamination and gel electrophoresis, quantitative results can be acquired by directly detecting change of fluorescence signals in the PCR amplification process through a photoelectric conduction system, result repetition is good, detection sensitivity is high, and detection on high-throughput samples can be achieved.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR primer combination for rapidly detecting Babesia canis, and also relates to a kit containing the primer combination and a detection method for Babesia canis, belonging to the technical field of biological detection. Background technique [0002] Babesiosis is a blood-borne protozoan disease that occurs widely in various livestock and is transmitted by ticks. It parasitizes in red blood cells singly or in pairs in domestic animals, causing severe anemia and hemoglobinuria. There are 5 kinds of pathogens that cause babesiosis in dogs: (i) Babesia veschneri (B.vogeli), which is mainly transmitted by Rhizocephalus haematocephalus, which exists not only in the tropics and subtropics, It is also distributed in cold regions; (ii) Babesia canis (B.canis), which is mainly transmitted by Derma ticks, can also be transmitted by Rhizocephalus blood red ticks, blood ticks, etc., only distributed in Europe; ( ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/90
CPCC12Q1/6851C12Q1/6893C12Q2531/113C12Q2563/107
Inventor 张才邵琦王臣谷文峰高志峰爨淑楠和俊豪陈小燕唐正露邵玉桃陈文彬刘彤
Owner 洛阳禹迹生物工程有限公司
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