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Primers, probes and kits for fluorescent quantitative PCR detection and typing of Babesia

A Babesia, fluorescence quantitative technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, etc., can solve the problem that molecular diagnostic technology can only detect a single or several Babesia species.

Inactive Publication Date: 2014-10-15
YANGZHOU UNIV
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AI Technical Summary

Problems solved by technology

[0003] In order to solve the problem that existing molecular diagnostic techniques can only detect one or several species of Babesia nucleic acid deficiency, the present invention provides a rapid, specific, high-sensitivity quantitative PCR that is suitable for clinical use to detect important species of Babesia Primers, probes and kits for nucleic acids

Method used

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  • Primers, probes and kits for fluorescent quantitative PCR detection and typing of Babesia
  • Primers, probes and kits for fluorescent quantitative PCR detection and typing of Babesia
  • Primers, probes and kits for fluorescent quantitative PCR detection and typing of Babesia

Examples

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Effect test

Embodiment 1

[0058] Example 1: PCR amplification of Babesia gibsoni positive strain

[0059] Whole blood was obtained from positive dogs infected with Babesia gibsoni (17 cases), and DNA was extracted with commercial kits and conventional methods, and 10 μl was used as a PCR template. The technique of the present invention is used for PCR amplification and high-resolution melting curve analysis (the specific technical details are as described above). The results showed that PCR could specifically and efficiently amplify all 17 positive strains of Babesia canis, and the melting temperatures of double peaks (63°C and 69°C) appeared.

Embodiment 2

[0060] Embodiment 2: the epidemiological investigation of dog infection Babesia

[0061] From the whole blood obtained from 372 dogs who came to the veterinary hospital for physical examination, vaccination, or medical treatment, DNA was extracted with commercial kits and conventional methods, and 10 μl was used as a PCR template. Carry out PCR amplification with the technology of the present invention (the specific technical details are as described above). The results showed that 22% of dogs (90 / 372) had Babesia infection. Only 6% of dogs infected with Babesia showed clinical symptoms, and 13% showed abnormal blood routine; while 81% of infected dogs were completely healthy, without clinical symptoms and abnormal blood routine. The PCR diagnostic results were confirmed by electrophoresis, sequencing and other tests.

[0062]

[0063]

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Abstract

The invention discloses a primer, a probe and a kit for fluorescence quantitative PCR (Polymerase Chain Reaction) detecting and typing of babesia. According to the invention, the primer sequence is as shown in SEQID (SEQuence IDentifier) NO.1, 2 and 3 and the probe is as shown in SEQIDNO.4 and 5. A typing method of the babesia comprises the following steps of: carrying out PCR amplification on the to-be-detected bacterium by using the primer and the probe to obtain Erich bacterium positive samples of 290bp (base pair) bands, wherein the fluorescence of the samples is enhanced at the wavelength of 640nm in fluorescence detection; and typing the babesia according to the melting temperature. The primer, the probe and the kit disclosed by the invention have obvious technical advantages, have very high specificity and sensitivity and can be used for conveniently detecting the babesia of important species.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR detection reagent and a detection method for the molecular diagnosis of Babesia. This invention effectively amplifies 21 important species of Babesia infecting dogs, cats, cattle, sheep, horses and humans (B.gibsoni, B.canis canis, B.canis rossi, B.canis vogli, B.felis, B. leo, B. bovis, B. equi, B. bigemina, B. caballi, B. capreoli, B. odocoilei, B. crassa, B. hongkangensis, B. vitalii, B. ovata, B. motasi, B. rodhaini, B.poelea, B.divergens, B.microti), without amplifying the nucleic acids of other similar blood parasites. Background technique [0002] Babesia spp. can cause a variety of diseases in animals and humans characterized by fever, anemia, and emaciation. Babesia is the second most common blood-borne parasitic disease infecting mammals worldwide. The existing laboratory diagnostic techniques for Babesia infection mainly include: 1) Observation of Babesia with blood pictures. The se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 王成明陶建平
Owner YANGZHOU UNIV
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