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Dermatophilus congolensis and Dermatophilaceae fluorescent quantitative PCR (polymerase chain reaction) primers, probes and kit

A fluorescent quantitative and primer-probe technology, which is applied in the determination/testing of microorganisms, microorganisms, and methods based on microorganisms, can solve the problems of insufficient specificity and sensitivity, time-consuming, etc., and achieve high sensitivity and high specificity.

Inactive Publication Date: 2014-02-12
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, culture and other traditional laboratory diagnostic methods are not specific, sensitive, and time-consuming

Method used

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  • Dermatophilus congolensis and Dermatophilaceae fluorescent quantitative PCR (polymerase chain reaction) primers, probes and kit
  • Dermatophilus congolensis and Dermatophilaceae fluorescent quantitative PCR (polymerase chain reaction) primers, probes and kit
  • Dermatophilus congolensis and Dermatophilaceae fluorescent quantitative PCR (polymerase chain reaction) primers, probes and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: the identification of D. congo standard strain

[0036] The laboratory isolated D. congo from the skin of sheep with dermatophilia. In this study, the bacteria were recovered, cultured and stained, and the bacteria showed characteristic colonies ( image 3 ) and staining ( Figure 4 )characteristic. Purified bacteria, DNA extraction, PCR amplification and high-resolution melting curve analysis using the technology of the present invention (specific technical details are as described above). The results showed that PCR can specifically and efficiently amplify the DNA of D. congo, and a melting temperature of 63°C appeared.

Embodiment 2

[0037] Example 2: PCR amplification and typing identification of clinical isolates

[0038] From 12 cows suffering from dermatophilia, 34 skin scabs were collected; each skin scab sample was divided into two, one was used for bacterial culture and staining identification, and the other was directly used for DNA extraction and PCR amplification. increase. The results showed that culture and staining identified 14 skin scab samples as positive (14 / 34, 42%), while the positive rate detected by PCR technique was 100%. The high-resolution melting curves showed that among the 34 strains of Dermatophilus, one strain was Dermatophilus cheloniformis, and the rest were Dermatophilus congo. Genetic sequencing confirmed the conclusions of high-resolution melting curves.

[0039]

[0040]

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Abstract

The invention relates to fluorescent quantitative PCR (polymerase chain reaction) primers, probes and a kit for Dermatophilus molecular diagnosis. The primer sequences are disclosed as SEQ ID NO.1, 2 and 3, and the probes are disclosed as SEQ ID NO.4 and 5. The primers and probes, and a PCR reaction solution constitute the fluorescent quantitative PCR detection kit. The primers and probes provided by the invention can be used for typing Dermatophilus congolensis and Dermatophilaceae. When being used for detecting Dermatophilus, the invention has the advantages of high speed, higher specificity and higher sensitivity.

Description

technical field [0001] The invention relates to fluorescent quantitative PCR detection primers, probes and kits for molecular diagnosis of dermatophilus. Background technique [0002] Dermatophilia is a skin disorder characterized by exudative and hyperplastic dermatitis, superficial epidermal necrosis, and scab formation. The disease primarily affects cattle, sheep, and horses; it can also affect goats, cats, dogs, many wild animals, and reptiles. Recently, there have been many reports of people contracting the disease around the world. [0003] Laboratory diagnosis of dermatophilus mainly relies on the observation of characteristic colonies of dermatophilus after culture and microscopic appearance of Gram-positive branched hyphae or train-track-shaped spores arranged in rows. However, culture and other traditional laboratory diagnostic methods are not specific, sensitive, and time-consuming. Therefore, it is particularly important and urgent to establish a new technolog...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/01
Inventor 王成明许传灵张继垒危蓝菁
Owner YANGZHOU UNIV