CN110923349B - Species-specific detection molecular tags 3283 and 3316 of yersinia enterocolitica and rapid detection method thereof - Google Patents
Species-specific detection molecular tags 3283 and 3316 of yersinia enterocolitica and rapid detection method thereof Download PDFInfo
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Abstract
The invention discloses two detection targets 3283/3316 of yersinia enterocolitica and detection primers thereof, wherein the sequences of the detection targets are shown as SEQ ID NO. 1 and SEQ ID NO. 2. The yersinia enterocolitica detection target disclosed by the invention can be used for quickly detecting the yersinia enterocolitica in samples such as food. The invention discloses a special detection primer, which specifically comprises the following components: 3283 tag forward primer 3283-F, 3283 tag reverse primer 3283-R;3316 tag Forward primer 3316-F, 3316 tag reverse primer 3316-R. And detecting by PCR amplification and agarose gel electrophoresis, and obtaining a detection result through an electrophoresis product result. The method can effectively detect the yersinia enterocolitica in samples such as food, has the advantages of rapidness, high efficiency, simple operation and the like, and can be applied to the fields of food, inspection and quarantine and the like.
Description
Technical Field
The invention belongs to the technical field of gene detection of yersinia enterocolitica, and particularly relates to species-specific detection molecular tags 3283 and 3316 of two yersinia enterocolitica and a rapid detection method thereof.
Background
Nucleic Acid Amplification Test (NAAT) is a rapid, easy-to-use, sensitive, economical and low-labor cost detection method for detecting pathogenic bacteria in food and clinical environments, which is gradually emerging in recent years. Gastrointestinal multiplex panels (GIMPs) designed based on this method are also increasingly used in clinical tests.
Yersinia enterocolitica (Ye) is a gram-negative pathogenic bacterium widely existing in the world and can cause Yersinia disease which is common to human and livestock. The treatment or consumption of uncooked pork is a major cause of illness in humans. Patients with yersinia disease often present with clinical syndromes consisting of fever, abdominal pain, and diarrhea; acute yersinia may cause serious complications such as sepsis, which are easily present in immunocompromised hosts such as infants and patients receiving immunosuppressive therapy. The pathogenic bacteria of the yersinia disease are widely distributed, so that the number of the yersinia disease is more, and according to the recent reports of European Union, the number of the yersinia disease is the third to suffer from the diseases of people and livestock for many years. Monitoring data from the food-borne disease active monitoring network (FoodNet) operated by the american centers for disease control and prevention (CDC) show that the incidence of yersinia enterocolitica infection has increased over the last four years (2015-2018) (139, 2016, 302, 2017, 489, 2019, 465). Therefore, timely detection of yersinia enterocolitica contamination in food products (particularly meat products) and prevention of the contaminated food products from entering the market are important means to reduce the incidence of yersinia disease.
Compared with other common food-borne pathogenic bacteria, the yersinia enterocolitica has the characteristics of slow growth and strong cold resistance, so that the separation and identification of the yersinia enterocolitica are performed by adopting a cold enrichment method for pretreatment. This results in a long separation and identification period (7-10 days) and a low detection rate of Yersinia enterocolitica. The high time and labor costs of detecting yersinia enterocolitica make it difficult to detect yersinia enterocolitica contamination in food products in a timely manner. In recent years, with the continuous development of genomics technology, more and more specific molecular tags are excavated and applied to the detection of specific strains. A method for amplifying a virulence gene ail by PCR is introduced into the latest revised pathogenic enterocolitis yersinia detection of ISO (ISO 10273. At the same time, the U.S. CDC also began to use GIMP to detect gastrointestinal pathogens and data showed that GIMP usage in the U.S. laboratory increased from <1% (2013, 2/460) to 14% (2016, 59/421).
Compared with the traditional culture method and biochemical identification, the PCR detection has the following advantages: (1) The economy is reasonable, the PCR detection cost is low, and a large amount of manpower and material resources can be saved; (2) The method has the potential of high flux, and PCR can be carried out in large batch and can be combined with new detection technologies such as a microfluidic chip and the like, so that the potential of high flux detection is realized; (3) The timeliness is strong, and the PCR detection can quickly obtain a detection result, so that the kit can be used for food safety monitoring or suspected case diagnosis. Therefore, the excavation of the yersinia enterocolitica species-specific molecular tag has important significance for the development of a rapid detection method of the yersinia enterocolitica and the perfection of a food monitoring network.
Disclosure of Invention
The invention aims to obtain two molecular labels 3283 and 3316 specific to enterocolitis yersinia species by comparison and search aiming at the current situation that the molecular labels for molecular detection of enterocolitis yersinia are mostly virulence genes and the molecular labels for species specificity of enterocolitis yersinia are not available, and special PCR detection primers for the two molecular labels are designed for specific detection.
In order to achieve the purpose, the invention adopts the technical scheme that: a group of specific detection molecular tags 3283 and/or 3316 of yersinia enterocolitica, wherein the molecular tags are DNA fragments, and the molecular tags 3283 encode proteins with amino acid sequences shown as SEQ ID NO. 3; the molecular tag 3316 encodes a protein with an amino acid sequence shown in SEQ ID NO. 4.
Preferably, the molecular tag 3283 has a nucleotide sequence as shown in (a) or (b):
(a) 1, as shown in SEQ ID NO;
(b) Has at least 90 percent of homology with the nucleotide sequence shown in SEQ ID NO. 1, and encodes protein with the amino acid sequence shown in SEQ ID NO. 3.
Preferably, the molecular tag 3316 has a nucleotide sequence as shown in (c) or (d):
(c) A nucleotide sequence shown as SEQ ID NO. 2;
(d) Has at least 90 percent of homology with the nucleotide sequence shown in SEQ ID NO. 2, and encodes protein with the amino acid sequence shown in SEQ ID NO. 4.
The invention also provides application of the molecular tag 3283 or/and 3316 in preparation of a reagent for detecting yersinia enterocolitica.
The invention also provides a group of primers for detecting the molecular tag 3283 or/and 3316, wherein the nucleotide sequence of an upstream primer for detecting the molecular tag 3283 is shown as SEQ ID NO. 5, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 6; the nucleotide sequence of the upstream primer for detecting the molecular tag 3316 is shown as SEQ ID NO. 7, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 8.
The invention also provides a kit for detecting the yersinia enterocolitica, which comprises the nucleotide sequence shown in SEQ ID NO. 5-8.
The invention also provides a method for detecting yersinia enterocolitica, which comprises the following steps:
(1) Extracting DNA of a microorganism to be detected, and carrying out PCR amplification on the extracted DNA by using the specific primer of the invention as a template;
(2) The amplification product was analyzed by agarose gel electrophoresis, and it was judged that Yersinia enterocolitica was contained in the sample if a band of about 350bp (3283 tag) or 279bp (3316 tag) was present as a result of the electrophoresis, and it was judged that Yersinia enterocolitica was not contained in the sample if a band of the target size was not present as a result of the electrophoresis.
The PCR amplification method adopts a 25 mu L reaction system which comprises the following steps: 10 XPCR reaction buffer 2.5. Mu.L, 25mmol/L MgCl 2 2.0. Mu.L, 0.5. Mu.L of 10mmol/L (each) dNTPs, 0.5. Mu.L of 10. Mu. Mol/L primer, 1U of Taq enzyme, 0.5-1. Mu.L of DNA template, and sterilized double distilled water to make up the volume to 25. Mu.L.
Preferably, the PCR reaction conditions of the 3283 molecular tag are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 20s, 30 cycles in total, and extension at 72 ℃ for 5min.
Preferably, the PCR reaction conditions of the 3316 molecular tag are: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 62 ℃ for 30s, and extension at 72 ℃ for 20s for 30 cycles; extension at 72 ℃ for 5min.
Compared with the prior art, the invention has the following beneficial effects:
(1) Based on the whole genome sequencing information of the yersinia enterocolitica, the invention excavates the species specific molecular tag of the yersinia enterocolitica through pan-genome analysis, and adopts the PCR amplification technology for verification, so that the result is accurate and reliable.
(2) The PCR method for detecting the yersinia enterocolitica disclosed by the invention can be used for simultaneously detecting a plurality of specific molecular labels of the yersinia enterocolitica, can save a large amount of manpower and material resources compared with the traditional biochemical identification method, can simplify the experimental process, eliminates a large amount of false positive results, and provides possibility for high-throughput detection of the yersinia enterocolitica pollution condition in samples such as food and the like.
(3) The PCR method adopted by the invention can accurately know the yersinia enterocolitica pollution condition in the sample only by agarose gel electrophoresis for result analysis, does not need subsequent steps such as DNA sequence determination and the like, reduces the time and economic cost brought by the DNA sequence determination, and reduces the influence of the DNA sequence determination error on the result reliability.
(4) The molecular labels used for detecting the yersinia enterocolitica are mostly virulence factors, while the specific molecular labels aiming at the yersinia enterocolitica are rarely reported.
Drawings
FIG. 1 is a schematic diagram showing the results of detection of the presence of Yersinia enterocolitica molecular tag 3283 using 23 genomic DNAs of Yersinia enterocolitica as a template (in the figure, samples No. 1 to 23 are Yersinia enterocolitica; sample No. 24 is a blank control; the size of the target band is 358bp, and it is apparent from the figure that there are bands 1 to 23).
FIG. 2 is a diagram showing the results of detection of the presence of Yersinia enterocolitica molecular tag 3316 using 23 genomic DNAs of Yersinia enterocolitica strains as templates (in the figure, samples No. 1 to 23 are Yersinia enterocolitica; sample No. 24 is a blank control; the size of the target band is 279bp, and it is apparent from the figure that there are bands 1 to 23).
FIG. 3 is a schematic diagram showing the specific test result of Yersinia enterocolitica detection molecular tag 3283 in 32 strains of common pathogenic bacteria of non-Yersinia such as Bacillus cereus and Pseudomonas aeruginosa (in the diagram, samples No. 1 to 32 are other pathogenic bacteria except Yersinia, sample No. 33 is blank control, sample No. 34 is Yersinia enterocolitica, the size of the target band is 358bp, and no band exists in the diagram from 1 to 32, and band No. 34 is obvious).
FIG. 4 is a diagram showing the specific test result of the Yersinia enterocolitica detecting molecular tag 3316 in 32 common pathogenic bacteria of non-Yersinia such as Bacillus cereus and Pseudomonas aeruginosa (in the diagram, samples No. 1-32 are other pathogenic bacteria except Yersinia, sample No. 33 is blank control, sample No. 34 is Yersinia enterocolitica, the size of the target band is 279bp, and no band exists in the diagram from 1-32, and band No. 34 is obvious).
FIG. 5 is a diagram showing the specific test results of Yersinia enterocolitica detection molecule tag 3283 in Yersinia strains such as 37 Yersinia intermedia, yersinia freundii, yersinia ruckeri and Yersinia burkei (in the diagram, samples No. 1-37 are Yersinia furchii, intermediate Yersinia ruckeri and Yersinia burkei; sample No. 38 is Yersinia enterocolitica; sample No. 39 is a blank control; the size of the target band is 358 bp; no bands from 1 to 37 are shown in the diagram; and band No. 38 is evident).
FIG. 6 is a diagram showing the results of specific tests of the Yersinia enterocolitica-detecting molecular tag 3316 in 37 Yersinia intermediate strains, yersinia furiosa, yersinia ruckeri and Yersinia bursitis strains (samples No. 1-37 in the figure are Yersinia furiosa, intermediate, yersinia ruckeri and Yersinia bursitis; sample No. 38 is Yersinia enterocolitica; sample No. 39 is a blank control; the size of the target band is 279 bp; no bands from 1 to 37 in the figure; and the 38 band is evident).
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.
Example 1 search of Yersinia species-specific molecular signatures for enterocolitis
The screening of yersinia enterocolitica species-specific molecular signatures is mainly based on the pan-genomic analysis results. The genome-wide mapping data of 16 Yersinia enterocolitica strains (from NCBI database), 23 Yersinia enterocolitica strains (the strains are the strains stored in the experiment and the sequencing is completed in the laboratory), and 78 other Yersinia strains (the Yersinia enterocolitica strains, the types and the quantities are shown in Table 1) are selected for genome-wide analysis. The Pan-genome Analysis adopts an MP method in prokaryotic Pan-genome automated Analysis software (PGAP), and the Analysis result is processed by a local Perl script to obtain the core gene and non-core gene information of all strains. Then selecting genes specific to the yersinia enterocolitica, removing non-specific sequences through local Blast comparison, and obtaining final specific molecular tags 3283 (SEQ ID NO: 1) and 3316 (SEQ ID NO: 2) of the yersinia enterocolitica species after PCR amplification verification, wherein the nucleotide sequence of the protein coded by the molecular tag 3283 is shown in SEQ ID NO:3, and the nucleotide sequence of the protein coded by the molecular tag 3316 is shown in SEQ ID NO: 4.
Table 1: the species and number of strains used for pan-genomic analysis
Example 2 establishment of molecular target quick detection method for Yersinia enterocolitica
Specific amplification primers were designed based on the sequence molecular tags 3283 (SEQ ID NO: 1) and 3316 (SEQ ID NO: 2), and the sequences of the primers are shown in Table 2:
table 2: label 3283 and 3316 primer sequences
PCR amplification verification was performed using genomic DNA of 23 Yersinia enterocolitica strains isolated from food in this laboratory as a template.
The PCR system was 25. Mu.L, which included 2.5. Mu.L of 10 XPCR reaction buffer, 25mmol/L MgCl 2 2.0. Mu.L, 0.5. Mu.L of 10mmol/L (each) dNTPs, 0.5. Mu.L of 10. Mu. Mol/L primer, 1U of Taq enzyme, 0.5-1. Mu.L of DNA template, and sterile double distilled water to make up the volume to 25. Mu.L.
The PCR reaction conditions of the 3283 molecular tag are as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 20s for 30 cycles; extension at 72 ℃ for 5min.3316 PCR reaction conditions for the molecular tag were: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 62 ℃ for 30s, and extension at 72 ℃ for 20s for 30 cycles; extension at 72 ℃ for 5min.
The PCR results were checked by agarose gel electrophoresis (agarose concentration of the agarose gel used was 1.0%), and if the electrophoresis results showed 358bp (3283 molecular tag, FIG. 1) or/and 279bp (3316 molecular tag, FIG. 2) size bands, the strain was considered to be Yersinia enterocolitica (or Yersinia enterocolitica contained in the sample). If no band or a band of the correct size is present, it indicates that the strain is not yersinia enterocolitica (or that the sample does not contain yersinia enterocolitica). The results are shown in table 3 (fig. 1 and 2):
table 3: and (3) establishing an experimental result by using a detection method of 23 yersinia enterocolitica molecular targets 3283 and 3316.
Note: in the table, "-" indicates that the PCR amplification result was negative, and "+" indicates that the PCR amplification result was positive.
Example 3 result of evaluating specificity of Yersinia enterocolitica PCR detection method
Specific tests of the molecular tags 3283 and 3316 for Yersinia enterocolitica were carried out using 37 Yersinia strains of Yersinia enterocolitica such as Yersinia intermedia, yersinia freundii, yersinia ruckeri and Yersinia bursitis, and 32 Yersinia cerealis and Pseudomonas aeruginosa. Extracting the genome DNA of the corresponding strain as a template, performing PCR amplification according to the PCR amplification system and the amplification conditions described in example 1, and finally detecting the DNA by agarose gel electrophoresis. The detection results are shown in tables 4 and 5 (corresponding electrophoresis result pictures are respectively shown in fig. 3 and 4 and fig. 5 and 6).
Table 4: the specific test results of the molecular tags 3283 and 3316 in 32 strains of common pathogenic bacteria of non-Yersinia such as Bacillus cereus and Pseudomonas aeruginosa
Note: in the table, "-" indicates that the PCR amplification result was negative, and "+" indicates that the PCR amplification result was positive.
Table 5: results of specificity test of molecular tags 3283 and 3316 in 37 strains of Yersinia such as Yersinia intermedia, yersinia freundii, yersinia ruckeri, yersinia berkowii
Note: in the table, "-" indicates that the PCR amplification result was negative, and "+" indicates that the PCR amplification result was positive.
In conclusion, the molecular tags 3283 and 3316 of the present invention are specific to yersinia enterocolitica, which cannot be detected in common disease-treating species and yersinia enterocolitica, so the molecular tags 3283 and 3316 can be used as specific tags specifically for detecting yersinia enterocolitica. The primers for amplifying the two molecular tags can form a kit for detecting the yersinia enterocolitica.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> institute of microbiology, science institute of Guangdong province (center for microbiological analysis and detection in Guangdong province), kyork Biotech Co., ltd
<120> species-specific detection molecular tags 3283, 3316 of yersinia enterocolitica and rapid detection method thereof
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Claims (4)
1. The application of specific detection molecular tag 3283 of yersinia enterocolitica as a detected target in identifying the yersinia enterocolitica is characterized in that the nucleotide sequence of the molecular tag 3283 is shown as SEQ ID NO: 1; the molecular tag 3283 is the yersinia enterocolitica, and the application is the non-disease diagnosis and treatment purpose.
2. The application of the primer pair for detecting the molecular tag 3283 of the claim 1 in preparing the reagent for identifying the yersinia enterocolitica is characterized in that the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO. 5, and the nucleotide sequence of a downstream primer of the primer pair is shown as SEQ ID NO. 6; and if the amplification product of the primer pair has a strip with the size of 350bp, judging that the sample contains the yersinia enterocolitica, and if the amplification product of the primer pair does not have a strip with the size of 350bp, judging that the sample does not contain the yersinia enterocolitica.
3. A method for detecting yersinia enterocolitica for non-disease diagnostic and therapeutic purposes, comprising the steps of:
(1) Extracting DNA of a microorganism to be detected, and carrying out PCR amplification by using the extracted DNA as a template and the primer pair as claimed in claim 2;
(2) And (3) carrying out agarose gel electrophoresis analysis on the amplification product, judging that the sample contains the yersinia enterocolitica if a strip with the size of 350bp appears in an electrophoresis result, and judging that the sample does not contain the yersinia enterocolitica if the strip with the size of 350bp does not appear in the electrophoresis result.
4. The method of claim 3, wherein the PCR reaction conditions are: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 20s, 30 cycles in total, and extension at 72 ℃ for 5min.
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| CN101200760A (en) * | 2007-12-13 | 2008-06-18 | 中国检验检疫科学研究院 | Preparation and utilization method of yersinia genus rapid detection reagent kit |
| CN101748197A (en) * | 2008-12-12 | 2010-06-23 | 上海复星医药（集团）股份有限公司 | Target sequence for detection of yersinia enterocolitica and kit |
| CN103725752A (en) * | 2012-10-10 | 2014-04-16 | 苏州四同医药科技有限公司 | Method for highly-specific highly-sensitive detection of food-borne pathogenic yersinia |
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| CN103468811B (en) * | 2013-09-17 | 2014-09-10 | 北京卓诚惠生生物科技有限公司 | Yersinia enterocolitica virulence gene multiplex-PCR (Polymerase Chain Reaction) detection primer group and kit |
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