Fluorescent quantitation PCR detection of Ehrlichia bacterium and typing primer, probe and kit
A technology for fluorescence quantification and detection of primers, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve high specificity and sensitivity, obvious technical advantages, and good economic benefits.
Inactive Publication Date: 2013-04-24
YANGZHOU UNIV
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Problems solved by technology
[0004] In order to solve the deficiency that the existing molecular diagnostic technology can only detect a single type of Ehrlichia, the present invention provides a rapid, specific, high-sensitivity quantitative PCR method suitable for clinical use to detect all kinds of Ehrlichia Primers, Probes and Kits
Method used
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Embodiment 1
[0040] Example 1: Molecular Identification of Ehrlichia
[0041] Molecular diagnosis was performed on 10 dogs with Ehrlichia confirmed clinically, microscopically on blood smears, and serologically. The operating procedure was as above, and 400 μl of EDTA whole blood was used to extract DNA for PCR amplification. All 10 samples were PCR positive for Ehrlichia, with a characteristic melting temperature of 63-64°C (eg Figure 5 shown). Gene sequencing confirmed the results of PCR.
Embodiment 2
[0042] Example 2: Molecular epidemiological investigation of E. canis infection
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The invention discloses a fluorescent quantitation PCR detection of Ehrlichia bacterium and a typing primer, a probe and a kit. The sequence of the primer is shown in SEQ ID No. 1 and 2; the probe is shown in SEQ ID No. 3 and 4. The primer and the probe are adopted to carry out the PCR amplification for the bacterium to be detected and amplify 210bp band, and the fluorescent detection enhanced in 640 nanometers wavelength is positive samples of Ehrlichia bacterium; and then the Ehrlichia bacterium are typed according to the melting temperature. The invention has obvious technical advantages, high specificity and high sensitiveness, can conveniently detect all kinds of Ehrlichia bacteria, can be used for diagnosing and treating human and various animals (cattle, dogs and sheep) and Ehrlichia chafeensis diseases, and has good economic benefits.
Description
technical field [0001] The invention relates to primers, probes and kits for fluorescent quantitative PCR detection and typing of molecular diagnosis of Ehrlichia infection. Background technique [0002] Ehrlichia is an obligate intracellular parasite that lives in the cytoplasm of monocytes and neutrophils. Ehrlichiosis is transmitted by ticks and is widespread worldwide, causing diseases in humans and various animals (cattle, dogs, sheep) characterized by fever and anemia. The clinical diagnosis of the disease mainly relies on epidemiology and symptoms, combined with the detection of Ehrlichia by microscopic observation of blood smears. The sensitivity of clinical diagnosis is low, and the existing serological diagnostic methods cannot distinguish between infected recovered patients and clinically infected patients. [0003] Important species of Ehrlichia infecting humans and animals include: canine E. canis, human E. chafeensis and E. ewingii, bovine E. ruminatium, shee...
Claims
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Login to View More IPC IPC(8): C12Q1/68C12Q1/10C12N15/11
Inventor 王成明王志强许传灵
Owner YANGZHOU UNIV
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