Primers and probes for real-time fluorescent PCR detection of Cryptobacter pyogenes and detection method

A Cryptobacterium pyogenes, real-time fluorescence technology, applied in the biological field, can solve the problems of difficult to compare bands, lack of sensitivity, specificity, fast operability, convenient detection method, low sensitivity, etc., to avoid polluting the environment and Operator, fast detection and identification, and accurate detection results

Active Publication Date: 2017-01-04
黄埔出入境检验检疫局综合技术服务中心
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional bacterial culture and biochemical identification take a long time. Conventional PCR detection requires follow-up gel running, ethidium bromide staining and observation under ultraviolet light. There is no way to judge the result of amplification. When the concentration of bacteria or DNA is low, the bands are not clear, the sensitivity is not high, and it is difficult to compare the difference of the bands
[0006] For C. pyogenes, there is currently a lack of rapid and convenient detection methods with higher sensitivity, specificity, and operability that can avoid environmental pollution and endanger operators such as EB.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Primers and probes for real-time fluorescent PCR detection of Cryptobacter pyogenes and detection method
  • Primers and probes for real-time fluorescent PCR detection of Cryptobacter pyogenes and detection method
  • Primers and probes for real-time fluorescent PCR detection of Cryptobacter pyogenes and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Establishment of a real-time fluorescent PCR detection system for Cryptobacter pyogenes

[0035] Design primers and probes:

[0036] Through targeted analysis of C. pyogenes and a large number of experimental summaries, primers and probes were designed based on Primer Express 3.0 based on the PLO gene sequence of C. pyogenes on GenBank (accession number U84782). The sequences of the primers are:

[0037] Upstream primer: a.p.-817-F: 5'-CCGACATTCCCACATAGCGTTT-3'

[0038] Downstream primer: a.p.-884-R: 5'-TTGACTCCCCGATCTTTCAAA-3'

[0039] The sequence of the probe is:

[0040] a.p.-840-probe: 5'-FAM-CGGCCCGAATGTCACCGCA-TAMRA-3'.

[0041] The primers and probes can be synthesized by conventional methods in the art. Primers and probes used in this example

[0042] Both entrust Bao Biosynthesis.

[0043] The instrument used for the real-time fluorescent PCR detection in the embodiment of the present invention is a fluorescent quantitative PCR instrument ABI7...

Embodiment 2

[0051] Embodiment 2: specificity and sensitivity test of the inventive method

[0052] (1) Detection specificity analysis

[0053] The present invention adopts a large number of bacterial strains, respectively uses the detection method established by the present invention to carry out real-time fluorescent PCR amplification, and observes whether the experimental bacterial strains have S-shaped amplification curves. Table 1 lists some of the strains used in this experiment. Experiments have found that there is and only A. Pyogenes has exponential amplification, and no other bacteria have S-type amplification curves, which shows that the method established by the present invention for A. The TaqMan real-time fluorescent PCR method for Pyogenes gene detection has good specificity, see appendix figure 1 shown. attached figure 1 Among them, 1 is Cryptobacter pyogenes; 2-14 are Salmonella, Vibrio cholerae, Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus...

Embodiment 3

[0060] Embodiment 3: the detection of clinical separation sample

[0061] In this example, the established TaqMan real-time fluorescent PCR method was used to detect 24 disease materials, and API Coryne was used for isolation and identification. A. pyogenes was detected in 16 of the 24 disease materials, see appendix Figure 5 , the identification results are completely consistent. Through the comparison experiment of clinically isolated samples, it is found that adopting the fluorescent PCR method of the present invention can save a lot of detection time and ensure higher specificity and accuracy at the same time. The method of the invention is fast, accurate and reliable, and is suitable for rapid detection and identification of A. pyogenes. attached Figure 5 Among them, 1 is C. pyogenes strain; 2-17 are positive clinical samples; 18-24 are negative clinical samples.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
correlation coefficientaaaaaaaaaa
PCR efficiencyaaaaaaaaaa
Login to View More

Abstract

The invention discloses a primer, a probe and a detection method for real-time fluorescent PCR detection of Cryptobacterium pyogenes. The present invention provides a pair of specific primers for Cryptobacterium pyogenes, wherein the DNA sequence of the upstream primer is shown in SEQ ID NO.1; the DNA sequence of the downstream primer is shown in SEQ ID NO.2; at the same time, a probe is provided, the DNA sequence of which is As shown in SEQ ID NO.3. Based on the primers and probes, the present invention summarizes the scientific real-time fluorescent PCR detection system and precise reaction procedure conditions, and successfully establishes a real-time fluorescent PCR detection method for Cryptobacterium pyogenes, which is simple and safe to operate, and the minimum detection DNA concentration is 77.6 fg, the minimum detection bacteria concentration is 63CFU / mL, and the whole amplification process takes less than 50 minutes. It has higher sensitivity, specificity and operability, and can well meet the rapid detection and identification of Cryptobacter pyogenes in veterinary clinics.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, to a real-time fluorescent PCR detection primer, probe and detection method for Cryptobacter pyogenes TaqMan. Background technique [0002] Arcanobacterium pyogenes (A.pyogenes) is a pleomorphic, non-motile, Gram-positive bacillus that was once considered to be Corynebacterium pyogenes and was then classified into the genus Actinomyces. Ramos reclassified it to the genus Cryptobacter after performing phylogenetic analysis based on the 16S rRNA gene. Cryptobacterium pyogenes often parasitizes mucous membranes such as the upper respiratory tract and digestive tract. Such as pneumonia, mastitis, endometritis, arthritis, otitis externa, cystitis and liver, kidney abscess and other symptoms. [0003] Tang Jie et al. reported that the infection of musk deer with purulent cryptic rods can cause abscesses in the submandibular and nasal mirrors. And mixed with Escherichia coli, streptoco...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & AuthorityPatents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
Inventor刘二龙卢丽吕英姿蒋湘郑高彬秦焯敏林学勤林惠娇姚柏辉王定国
Owner黄埔出入境检验检疫局综合技术服务中心