Primers, probes, a test kit and a test method for triple real-time fluorescence PCR detection of four bacteria

A real-time fluorescence, primer detection technology, applied in biochemical equipment and methods, fluorescence/phosphorescence, microbial determination/inspection, etc. System amplification efficiency evaluation and other issues

Active Publication Date: 2013-04-24
许龙岩 +5
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

With the development of molecular biology techniques, there have been reports on the detection of Salmonella by ordinary PCR or real-time fluorescent PCR targeting invA, 16s rRNA, SpvC, invB, fimA, agfA, SEFl4, sefA, sdf, ssaQ, fimY, etc. as target genes , but the PCR method using the above sequence as the target fragment can only detect a part of the Sa serotype or its virulence gene, and cannot simultaneously obtain the serotype and toxin secretion type of the detected Salmonella.
At present, there is also the commercialized Bax system produced by DuPont. This system uses fluorescent PCR technology to detect Salmonella at a subordinate level. It has high specificity, but it cannot type Salmonella.
Camila et al. designed primers for ompC, SdfI, ViaB, and Spy as the target genes, established a multiplex PCR detection method for Salmonella (Salmonella spp), SE (Enteritidis), typhoid Sa (Typhi), ST (Typhimurium), and applied it to chicken For the detection of Salmonella, KIM et al. also designed multi

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  • Primers, probes, a test kit and a test method for triple real-time fluorescence PCR detection of four bacteria
  • Primers, probes, a test kit and a test method for triple real-time fluorescence PCR detection of four bacteria
  • Primers, probes, a test kit and a test method for triple real-time fluorescence PCR detection of four bacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0092] The selection of embodiment 1 strain and the design of detection primers and detection probes

[0093] A total of 142 strains of 34 different serotypes of Salmonella were selected, including 1 standard strain of Salmonella paratyphi C (IQCC10527), 21 isolates of Salmonella paratyphi C (SP1-SP21), and 1 standard strain of Salmonella choleraesuis (IQCC10502). , 24 isolates of Salmonella choleraesuis (SC1-SC24), 1 standard strain of Salmonella typhi (CMCC50071), 30 isolates of Salmonella typhi (ST1-ST30), 1 standard strain of Salmonella gallinarum typhi (CMCC50770), Salmonella typhi 33 strains (SG1-SG33) and 30 strains of other serotypes of Salmonella were isolated. In addition, 21 strains of non-Salmonella such as Proteus were selected. The strain information is shown in Table 1. The above strains were confirmed by API20E reagent strips and serological tests. The above-mentioned strains come from the China General Microorganism Culture Collection Management Center, the G...

Embodiment 2

[0122] Embodiment 2: Preparation of template DNA

[0123] The 22 strains of Salmonella paratyphi C, 25 strains of Salmonella choleraesuis, 31 strains of Salmonella typhi, 34 strains of Salmonella gallinarum typhi, 30 strains of other serotypes of Salmonella and 21 strains of non-Salmonella were cultured in buffered peptone water (BP) at 37°C for 10 hours , then take 10ml of the above-mentioned cultured bacteria solution and inoculate them into 90ml of TTB enrichment solution, incubate at 44.5°C for 18 hours, then take 1ml of the above-mentioned cultured bacteria suspension and transfer them into centrifuge tubes, centrifuge at 12000r / min for 5min to remove the supernatant, and use 1ml of deionized The water floats and precipitates, and then centrifuges at 12000r / min for 3min to remove the supernatant, repeats twice, and finally adds 200μl deionized water, and extracts DNA on a nucleic acid extractor for real-time fluorescent PCR amplification.

Embodiment 3

[0124] Embodiment 3: detection primer and detection probe specificity test

[0125] Single real-time fluorescent PCR reaction system 30μl, including: template DNA 1μl, 10×TaqMan buffer 4μl, 5mmol / LMgCl 2 2 μl, 2.5 mmol / L dNTPs 3 μl, 20 μmol / l detection probe 1 μl, 20 μmol / l detection primers 1 μl (total 2 μl), 0.55U UNG enzyme 0.2 μl, 2.5U / μl Taq polymerase 3 μl, deionized water 13.8 μl. The fluorescent PCR reaction parameters were 95°C for 30s, 95°C for 5s, 60°C for 34s, and 40 cycles.

[0126] 1. Specificity test 1: Specificity test of Salmonella choleraesuis and Salmonella paratyphi C

[0127] Extract the DNA of 25 strains of Salmonella choleraesuis, 22 strains of Salmonella paratyphi C, 31 strains of Salmonella typhi, 34 strains of Salmonella gallinarum typhi, 30 strains of other serotype Salmonella and 21 strains of non-Salmonella according to the method described in Example 2, according to the above A single real-time fluorescent PCR reaction system is used for real-ti...

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Abstract

The invention discloses primers, probes, a test kit and a test method for triple real-time fluorescence PCR detection of Salmonella choleraesuis, Salmonella paratyphi, Salmonella typhi, and Salmonella gallinarum. According to the present invention, a triple real-time PCR method is used, wherein a consensus sequence of Salmonella choleraesuis and Salmonella paratyphi is used for the detection of Salmonella choleraesuis and Salmonella paratyphi, a specific sequence of Salmonella typhi is used for the detection of Salmonella typhi, and a specific sequence of Salmonella gallinarum is used for the detection of Salmonella gallinarum. Whether a sample is contaminated by Salmonella choleraesuis, Salmonella paratyphi, Salmonella typhi, and Salmonella gallinarum is determined via a real-time fluorescence PCR amplification, and then Salmonella choleraesuis and Salmonella paratyphi are distinguished via a single fluorescent PCR amplification; the detection is rapid, the process of preparing sample and issuing test results can be finished in 31hours, the results are reliable, and sensitivity and specificity are high.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to triple real-time fluorescent PCR detection primers for Salmonella choleraesuis, Salmonella paratyphi C, Salmonella typhi and Salmonella gallinarum, Probes, detection kits and detection methods. Background technique [0002] Salmonella (Salmonella) belongs to the Enterobacteriaceae family and is an important intestinal pathogenic bacterium for humans. It can also reproduce or cause disease in the intestinal tract of animals. At present, there are more than 2,500 serotypes of Salmonella known, and more than 200 serotypes have been found in my country. There are two main categories of illnesses caused by Salmonella. One is typhoid and paratyphoid, which are caused by typhoid or paratyphoid Salmonella, and occasionally typhoid-like infections are caused by other Salmonella, such as Salmonella choleraesuis and Salmonella enteritidis. Salmonella gallinarum (Salmonella gallina...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12N15/11G01N21/64
CPCY02A50/30
Inventor 许龙岩袁慕云曹际娟柯碧霞相大鹏柯昌文
Owner 许龙岩
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