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Primer combinations and detection kits for identifying 33 serotypes of Streptococcus suis

A detection kit and primer combination technology, applied in the field of molecular biology, can solve the problems of incomplete understanding of information, time-consuming and laborious, molecular typing methods have not been implemented into detection systems, etc., and achieve optimized detection procedures, simplified detection steps, and good market. The effect of the application foreground

Inactive Publication Date: 2018-08-07
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of understanding of the sequence information of the other 18 types, this molecular typing method has not yet been developed into a complete detection system
Due to the incomplete understanding of the genome information of each serotype in previous studies, the identification of serotype-specific genes can only be determined by the method of southern hybridization. This method will also be affected by some experimental factors, and it is time-consuming and laborious.

Method used

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  • Primer combinations and detection kits for identifying 33 serotypes of Streptococcus suis
  • Primer combinations and detection kits for identifying 33 serotypes of Streptococcus suis
  • Primer combinations and detection kits for identifying 33 serotypes of Streptococcus suis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Determination of 33 serotype-specific genes of Streptococcus suis and the design of primers

[0029] 1. The 33 reference strains were obtained from the International Reference Laboratory of Streptococcus suis strains (the laboratory of Professor Marcelo Gottschalk, University of Montreal, Canada) for sequencing. The strains were cultured overnight at 37°C on a Columbia plate, and single-clonal colonies were picked and inoculated in THB medium, and cultured with shaking at 37°C for 8 hours.

[0030]2. Whole genome sequencing and interception of cps gene cluster. Genomic DNA was extracted and purified using Wizard Genomic DNA Purification kit (Promega, Madison, USA). After sequencing the strain genome with Illumina Solexa GAIIx (Illumina, San Diego, CA), a paired-end (PE) library with an average insert length of 500bp-2000bp was constructed, and the data was assembled with SOAP denovo (Release1.04). The cps gene clusters of each serotype were intercepted from ...

Embodiment 2

[0038] Embodiment 2 detects the establishment of 33 kinds of serotypes of Streptococcus suis mPCR method

[0039] Using the primers of Example 1, using the genomic DNA of 33 serotype reference bacterial strains of Streptococcus suis in Example 1 as a template, and double-distilled water without nucleic acid as a negative control, first carry out single-plex PCR amplification to each pair of primers, Check its specificity. It was found that in single-plex PCR, the serotypes corresponding to each pair of primers had 100% specificity, and then a multiplex PCR method was established to detect 33 serotypes of Streptococcus suis.

[0040] Considering the practicability and ease of operation, this embodiment divides the multiplex PCR method into three detection systems A, B, and C. System A is used to detect the most common serotypes in clinical isolates, 1-10, 14 and Type 1 / 2; System B detects types 11-21 (except type 14); system C detects types 22-31 and 33. The DNA of 33 referen...

Embodiment 3

[0042] The specificity verification of the mPCR detection method of 33 kinds of serotypes of Streptococcus suis of embodiment 3

[0043] With 33 reference strains of Streptococcus suis as detection objects, the specificity of the mPCR method of the present invention is detected in conjunction with the following strains: Klebsiella pneumoniae (Klebsiella pneumoniae) 46117-3 and other Streptococcus genus including 6 strains of Streptococcus pneumoniae ) ATCC700657, ATCC700670, ATCC700676, ATCC700902, ATCC700906 and ATCC49619, Streptococcus pyogenes 32003, Streptococcus sanguis 32214, Enterococcus faecalis 32221, 3 Streptococcus bovis, 3 Streptococcus bovis Streptococcus bovis ATCC33317, Streptococcus lutetiensis 033 and 2 strains of Streptococcus orisratti originally considered to be Streptococcus suis 32 (strain EA1172.91) and 34 (strain92-2742) types.

[0044] However, there was no non-specific amplification band in the detection of 1 strain of Klebsiella pneumoniae (Klebsiell...

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Abstract

The present invention provides a primer combination for identifying 33 serotypes of Streptococcus suis, the nucleotide sequence of which is shown in SEQ ID NO.1-62, using the multiplex PCR (mPCR) method, using the primer combination provided by the present invention, It can quickly and accurately detect whether the bacteria to be tested belong to Streptococcus suis and which serotype of Streptococcus suis it belongs to. The invention also provides a kit for identifying 33 serotypes of Streptococcus suis. The detection method and the kit thereof of the present invention have the advantages of accurate detection, high sensitivity, strong specificity, simplicity and speed, and good clinical specimen detection ability.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a combination of primers and a multiplex PCR (mPCR) detection method for detecting 33 serotypes of Streptococcus suis, and also relates to a method for detecting 33 serotypes of Streptococcus suis using the primers Reagent test kit. Background technique [0002] Streptococcus suis is one of the most important pathogens causing swine diseases worldwide, and it is also a zoonotic pathogen. The main route of human infection with Streptococcus suis is contact with dead pigs, the pathogenic bacteria invade through damaged skin and mucous membranes, or ingest uncooked sick and dead pork. Clinically, it is mainly divided into two types, namely, sepsis type and meningitis type. In addition, Streptococcus suis can also invade the joints, eyes and heart of the human body, causing suppurative arthritis, endophthalmitis, endocarditis and shock. In recent years, reports of human S. suis in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/14C12N15/11
Inventor 刘志杰郑翰白雪梅纪少博徐建国
Owner ICDC CHINA CDC
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