CN106434898B - Method, primer and kit for rapidly detecting yersinia pseudotuberculosis at constant temperature - Google Patents

Method, primer and kit for rapidly detecting yersinia pseudotuberculosis at constant temperature Download PDF

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CN106434898B
CN106434898B CN201610780457.8A CN201610780457A CN106434898B CN 106434898 B CN106434898 B CN 106434898B CN 201610780457 A CN201610780457 A CN 201610780457A CN 106434898 B CN106434898 B CN 106434898B
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primer
yersinia pseudotuberculosis
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downstream
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CN106434898A (en
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李园园
李雪玲
韦朝春
贾犇
刘伟
陆长德
李亦学
曹永梅
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Shanghai Wang Wang Food Group Co Ltd
Shanghai Industrial Institute For Research And Technology
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Shanghai Industrial Institute For Research And Technology
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Abstract

The invention discloses a method, a primer group and a kit for rapidly detecting yersinia pseudotuberculosis at constant temperature. The method comprises the following steps: extracting genome DNA from a sample to be detected; carrying out constant-temperature amplification reaction in an enzyme reaction system by taking the genome DNA as a template and a primer group capable of amplifying the specific sequence of the Yersinia pseudotuberculosis as a primer; and determining whether the sample to be detected has the yersinia pseudotuberculosis or not by judging whether the reaction result is positive or not. The detection method has the advantages of high sensitivity and high specificity, short detection time, simple result judgment, convenient operation, low cost and wide application prospect.

Description

Method, primer and kit for rapidly detecting yersinia pseudotuberculosis at constant temperature
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method, primers and a kit for rapidly detecting yersinia pseudotuberculosis at a constant temperature.
Background
Yersinia pseudotuberculosis (Yersinia pseudotuberculosis) belongs to Yersinia genus of Enterobacteriaceae, is an intestinal pathogenic bacterium which is co-infected with human and livestock, and can live in a low-temperature environment, so that refrigerator-stored food is an important infection source of the bacterium infection in modern society, and can cause gastrointestinal symptoms, mesenteric lymphadenitis and the like. The bacterial infection is mainly sporadic in the population and occasionally causes outbreaks of different scales. Because the clinical symptoms are not typical, the diagnosis is not clear and even misdiagnosis is easy to cause to delay treatment. Therefore, it is very important to prevent and detect the bacteria.
The traditional detection method for the yersinia pseudotuberculosis has the defects of long detection period, relatively complex operation and low detection efficiency, and is difficult to meet the requirements of high flux, high sensitivity, high specificity, rapidness and convenience in the detection process of the food-borne pathogenic bacteria in the modern society. In recent years, researchers have developed detection means such as PCR with the development of nucleic acid molecule detection technology, but this method requires a special detection instrument, and is not suitable for real-time in-situ detection widely used in the basic detection department, particularly in the production line of enterprises. In order to ensure food safety, a rapid, simple and accurate method for detecting yersinia pseudotuberculosis in food is urgently needed.
Loop-mediated isothermal amplification (LAMP) is a novel isothermal Nucleic acid amplification method developed in recent years, which designs 4 specific primers (including upstream and downstream outer primers F3 and B3, and upstream and downstream inner primers FIP and BIP, wherein FIP is composed of F1C and F2, and BIP is composed of B1C and B2) for 6 regions of a target sequence, and completes the Nucleic acid amplification reaction by incubating for about 60min at an isothermal condition, and generates a visible reaction by-product, white magnesium pyrophosphate precipitate (see Notomi T, OkayamaH, Masubuchi H, Yonekawa T, Watanabe K, Nuino N, Hase T. loop-mediated isothermal amplification reaction (2000, J8512; 63). The technology can be completed at a constant temperature without a PCR instrument or a fluorescent quantitative PCR instrument, can judge the reaction result by naked eyes, and has the advantages of high sensitivity, strong specificity, short reaction time, convenient operation, low cost and the like.
Primer design is the most critical step in LAMP technology, and the conventional method is to introduce the acknowledged specific gene of a certain organism to be detected into an online website (http:// primer explorer. jp/e) designed by LAMP primers, and set relevant parameters to generate a primer group. That is, the user must first ensure that the target gene is a specific sequence of the species to be tested. Taking the invention patents CN101182575B and CN 101200760A as examples, the detection of Yersinia pseudotuberculosis is carried out by LAMP technology aiming at specific sequences of Yersinia pseudotuberculosis, namely 16S-23S interval and gyrB gene, reported in literature. However, the so-called "recognized specific genes" are often based on a delayed knowledge and are not necessarily updated based on the ever-increasing genome data of microorganisms, so that primers obtained based on the target gene sequences are not necessarily able to ensure their specificity and/or versatility in practical use. The present invention, as shown in Table 1, shows the problems of insufficient primer specificity and unsuitability of versatility in the prior art. That is, the yersinia pseudotuberculosis detection sequence used in the prior art method is not actually specific to yersinia pseudotuberculosis, that is, it is possible that yersinia pseudotuberculosis is erroneously identified as yersinia pseudotuberculosis. Similar problems exist in the confirmation of the versatility, i.e., the possibility of missing part of the strains of Yersinia pseudotuberculosis. Therefore, a Yersinia pseudotuberculosis detection method capable of ensuring specificity and universality is urgently needed in the industry, and meanwhile, the requirements of basic detection departments on rapidness and convenience are met, and real-time on-site detection can be conveniently developed in an enterprise production line.
Disclosure of Invention
The invention aims to overcome the defects of insufficient primer universality and specificity in the primer design of the LAMP technology, fully utilizes abundant microbial genome sequence information in the current public data resources and corresponding sequence analysis tools, designs a primer group for specifically identifying the Yersinia pseudotuberculosis, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention designs Yersinia pseudotuberculosis LAMP primers based on microbial genome data resources (data 8/5/2013) in a GenBank database, and provides a method, a primer group and a kit for rapid isothermal amplification detection of Yersinia pseudotuberculosis. The detection method for detecting the yersinia pseudotuberculosis has the advantages of high sensitivity and specificity, short detection time, simple result judgment, convenience in operation and low cost.
The invention provides a method for rapidly detecting Yersinia pseudotuberculosis strains, which comprises the following steps:
(1) extracting genome DNA from a sample to be detected;
(2) carrying out constant-temperature amplification reaction under an enzyme reaction system by taking the genome DNA as a template and a primer group capable of amplifying the specific base sequence of the Yersinia pseudotuberculosis genome as a primer;
(3) and determining whether the sample to be detected has the yersinia pseudotuberculosis or not by judging whether the reaction result is positive or not.
The method for detecting the yersinia pseudotuberculosis strain at constant temperature extracts genome DNA from a sample to be detected, takes the genome DNA as a template and takes a yersinia pseudotuberculosis specific amplification primer group as a primer to carry out constant-temperature amplification reaction, and then determines whether the yersinia pseudotuberculosis exists in the sample to be detected by judging whether the reaction result is positive or not. Wherein, the enzyme reaction system includes but is not limited to DNA polymerase reaction system.
In the invention, the genome-specific alkali sequence of the Yersinia pseudotuberculosis is the sequence of 768567-768785 bp of the Yersinia pseudotuberculosis with GI number of 153946813.
In the present invention, the primer set capable of amplifying the Yersinia pseudotuberculosis genome-specific nucleotide sequence is a part of the nucleic acid sequence of 768567 to 768785bp of the genome (GI No. 153946813) or a part of the complementary strand thereof. Wherein the Yersinia pseudotuberculosis genome-specific base sequence refers to a base sequence that is unique to the Yersinia pseudotuberculosis genome and is not contained in the genomes of other microorganisms.
Wherein the primer group capable of amplifying the Yersinia pseudotuberculosis genome-specific base sequence includes, but is not limited to, primer group A, or any one selected from the primer group having a homology of 50% or more with a single sequence in the sequence of the primer group or the complementary strand sequence thereof.
Primer set a:
upstream outer primer F3_ a: 5'-GTTGTTGTATGAATTGCTGG-3' (SEQ ID NO: 1);
downstream outer primer B3_ a: 5'-GCGGATAACTCCTGTTCT-3' (SEQ ID NO: 2);
upstream inner primer FIP _ A: 5'-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3'
(SEQ ID NO：3)；
The downstream inner primer BIP _ A: 5'-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3'
(SEQ ID NO：4)。
In the present invention, the primer set capable of amplifying the specific base sequence of the yersinia pseudotuberculosis genome may further include a primer set having a homology of 50% or more with a single sequence in the aforementioned sequences of each primer set or the complementary strand sequence thereof, and the primer set includes, but is not limited to, the following primer set B:
primer set B:
upstream outer primer F3_ B: 5'-GAATTGCTGGAAATGGTTG-3' (SEQ ID NO: 5) (50% homology to primer F3_ A5'-GTTGTTGTATGAATTGCTGG-3');
downstream outer primer B3_ B: 5'-GCGGATAACTCCTGTTCT-3' (SEQ ID NO: 6);
upstream inner primer FIP _ B: 5'-GTAACGCTGAAATACCGAACTGTATGAAGGTGGTTTGAAACG-3'
(SEQ ID NO：7)；
The downstream inner primer BIP _ B: 5'-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3'
(SEQ ID NO：8)。
In the method of the present invention, the primer set capable of amplifying a Yersinia pseudotuberculosis genome-specific base sequence may or may not comprise a loop primer. The loop primer may be one or more, including primers LF and/or LB. The primer group capable of amplifying the specific base sequence of the Yersinia pseudotuberculosis genome is selected from any one of the following primer groups A 'and B'; or any one selected from the group consisting of primers having a homology of 50% or more with respect to the sequences of the primer groups A ', B' or the complementary strand sequences thereof:
primer set a':
upstream outer primer F3_ a: 5'-GTTGTTGTATGAATTGCTGG-3', respectively;
downstream outer primer B3_ a: 5'-GCGGATAACTCCTGTTCT-3', respectively;
upstream inner primer FIP _ A: 5'-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3', respectively;
the downstream inner primer BIP _ A: 5'-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3', respectively;
upstream loop primer LF _ a: 5'-TATGCTCATCCAGGCGTTTC-3' (SEQ ID NO: 9);
and/or, the downstream loop primer LB _ A: 5'-TGATGGCGATGGGGAAAATT-3' (SEQ ID NO: 10);
a primer set B':
upstream outer primer F3_ B: 5'-GAATTGCTGGAAATGGTTG-3', respectively;
downstream outer primer B3_ B: 5'-GCGGATAACTCCTGTTCT-3', respectively;
upstream inner primer FIP _ B: 5'-GTAACGCTGAAATACCGAACTGTATGAAGGTGGTTTGAAACG-3', respectively;
the downstream inner primer BIP _ B: 5'-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3', respectively;
upstream loop primer LF _ B: 5'-GGGATTTCAATATGCTCATCCAG-3' (SEQ ID NO: 11);
and/or, the downstream loop primer LB _ B: 5'-TGATGGCGATGGGGAAAATT-3' (SEQ ID NO: 12).
In specific embodiments, for example, the primer sets a 'and B' may comprise only one forward loop primer, only one downstream loop primer, or both an upstream loop primer and a downstream loop primer.
In a specific embodiment (including a loop primer), the enzyme reaction system for isothermal amplification is as follows: 1 XBstDNA polymerase reaction buffer, 2-9mmol/L Mg2+(MgSO4Or MgCl2) 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.4-1.0 mu mol/L LF and/or LB primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. In another embodiment (without loop primer), the enzyme reaction system for isothermal amplification is: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg2+(MgSO4Or MgCl2) 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. The loop primer contributes to the improvement of the reaction efficiency. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4，0.1％Triton X-100，2mMMgSO4. MgSO in 1 XBst DNA polymerase reaction buffer4And magnesium ion Mg in enzyme reaction system2+And (6) merging.
In the method, the reaction procedure of the constant-temperature amplification reaction is incubation at ① 60-65 ℃ for 10-90 min, preferably 10-60 min, and termination reaction at ② 80 ℃ for 2-20 min.
In the method of the present invention, the detection method includes, but is not limited to, electrophoresis detection, turbidity detection, color detection, or the like. The electrophoresis detection is preferably a gel electrophoresis detection method, and may be agarose gel or polyacrylamide gel. In the electrophoresis detection result, if the electrophoresis chart shows a characteristic step-shaped strip, the sample to be detected is positive to the Yersinia pseudotuberculosis and contains the Yersinia pseudotuberculosis; and if the electrophoretogram does not present a characteristic step-shaped strip, the sample to be detected is negative to the Yersinia pseudotuberculosis. The turbidity detection is to detect turbidity by visual observation or a turbidity meter, and if the detection tube is obviously turbid, the sample to be detected is positive to the yersinia pseudotuberculosis and contains the yersinia pseudotuberculosis; if no turbidity is found, the sample to be detected is negative to the Yersinia pseudotuberculosis. Or the bottom of the reaction tube can be visually observed whether the precipitate exists or not after centrifugation, if the precipitate exists at the bottom of the reaction tube, the sample to be detected is positive to the Yersinia pseudotuberculosis and contains the Yersinia pseudotuberculosis; if no precipitate is formed at the bottom of the reaction tube, the sample to be detected is negative to the Yersinia pseudotuberculosis.
The color development detection is to add color development reagent, including but not limited to calcein (50 μ M) or SYBRGreenI (30-50X), or hydroxynaphthol blue (i.e. HNB, 120-. When calcein or SYBR Green I is used as a color developing agent, if the color is orange after reaction, the sample to be detected is negative to Yersinia pseudotuberculosis; if the color after the reaction is green, the sample to be detected is positive to the Yersinia pseudotuberculosis and contains the Yersinia pseudotuberculosis. When hydroxyl naphthol blue is used as a color developing agent, if the color after reaction is violet, the sample to be detected is negative to Yersinia pseudotuberculosis; and if the color after the reaction is sky blue, the sample to be detected is positive to the Yersinia pseudotuberculosis. The chromogenic detection can be used for detecting the reaction result in real time or at the end point through a detection instrument besides observing the reaction result through naked eyes, and by reasonably setting the threshold value of the negative reaction, when the reaction result of the sample to be detected is lower than or equal to the threshold value, the sample to be detected is negative to the yersinia pseudotuberculosis; and when the reaction result of the sample to be detected is greater than the threshold value, determining that the sample to be detected is positive in Yersinia pseudotuberculosis. The detection instrument comprises but is not limited to a fluorescence spectrophotometer, a fluorescence quantitative PCR instrument, a constant temperature amplification microfluidic chip nucleic acid analyzer, a Genie II isothermal amplification fluorescence detection system and the like.
In the color development detection, if calcein or hydroxynaphthol blue is used as a color developing agent, the color developing agent can be added before the constant-temperature amplification reaction, or can be added after the constant-temperature amplification reaction is completed, preferably before the constant-temperature amplification reaction, so that the possibility of reaction pollution can be effectively reduced. If SYBR Green I is adopted as a color developing agent, the SYBR Green I is added after the isothermal amplification reaction is finished. If calcein is used as color-developing agent, 50 μ M calcein is added into enzyme reaction system, and 0.6-1mM [ Mn ] is added2+]For example, 0.6-1mM MnCl2。
The invention also provides a primer used in the method for detecting the Yersinia pseudotuberculosis strain at constant temperature. The primer comprises a primer group capable of amplifying specific base sequences of the Yersinia pseudotuberculosis genome, and the sequence of the primer is part of the nucleic acid sequence with the position of 768567-768785 bp of the Yersinia pseudotuberculosis genome with the GI number of 153946813 or part of the complementary strand of the Yersinia pseudotuberculosis genome.
Wherein the primer group capable of amplifying the Yersinia pseudotuberculosis genome-specific base sequence is selected from any one of the following primer groups, or from any one of the primer groups having a homology of 50% or more with a single sequence in the sequences of the primer groups or the complementary strand sequences thereof. Wherein, the primer group includes but is not limited to the following primer group A. The primer set having a homology of 50% or more with a single sequence in the aforementioned primer set sequence or its complementary strand sequence includes, but is not limited to, the following primer set B.
Primer set a:
upstream outer primer F3_ a: 5'-GTTGTTGTATGAATTGCTGG-3', respectively;
downstream outer primer B3_ a: 5'-GCGGATAACTCCTGTTCT-3', respectively;
upstream inner primer FIP _ A: 5'-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3', respectively;
the downstream inner primer BIP _ A: 5'-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3' are provided.
Primer set B:
upstream outer primer F3_ B: 5'-GAATTGCTGGAAATGGTTG-3', respectively;
downstream outer primer B3_ B: 5'-GCGGATAACTCCTGTTCT-3', respectively;
upstream inner primer FIP _ B: 5'-GTAACGCTGAAATACCGAACTGTATGAAGGTGGTTTGAAACG-3', respectively;
the downstream inner primer BIP _ B: 5'-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3' are provided.
In the primers used in the method for detecting Yersinia pseudotuberculosis at constant temperature, the primer group capable of amplifying Yersinia pseudotuberculosis genome-specific base sequences may or may not comprise one or more loop primers; the loop primer is LF and/or LB. The primer group capable of amplifying the specific base sequence of the Yersinia pseudotuberculosis genome is selected from any one of the following primer groups A 'and B'; or any one selected from the group consisting of primers having a homology of 50% or more with respect to the sequences of the primer groups A ', B' or the complementary strand sequences thereof:
primer set a':
upstream outer primer F3_ a: 5'-GTTGTTGTATGAATTGCTGG-3', respectively;
downstream outer primer B3_ a: 5'-GCGGATAACTCCTGTTCT-3', respectively;
upstream inner primer FIP _ A: 5'-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3', respectively;
the downstream inner primer BIP _ A: 5'-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3', respectively;
upstream loop primer LF _ a: 5'-TATGCTCATCCAGGCGTTTC-3', respectively;
and/or, the downstream loop primer LB _ A: 5'-TGATGGCGATGGGGAAAATT-3', respectively;
a primer set B':
upstream outer primer F3_ B: 5'-GAATTGCTGGAAATGGTTG-3', respectively;
downstream outer primer B3_ B: 5'-GCGGATAACTCCTGTTCT-3', respectively;
upstream inner primer FIP _ B: 5'-GTAACGCTGAAATACCGAACTGTATGAAGGTGGTTTGAAACG-3', respectively;
the downstream inner primer BIP _ B: 5'-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3', respectively;
upstream loop primer LF _ B: 5'-GGGATTTCAATATGCTCATCCAG-3', respectively;
and/or, the downstream loop primer LB _ B: 5'-TGATGGCGATGGGGAAAATT-3' are provided.
In a specific embodiment, the primer sets a 'and B' may comprise only one upstream loop primer, only one downstream loop primer, or both an upstream loop primer and a downstream loop primer. In a specific embodiment, the primers are respectively FIP, BIP, F3, B3, LF and LB primers or primers with homology of 50% or more with the above primer sequence or single primer in the complementary strand sequence.
The invention also provides a kit used in the method for detecting the Yersinia pseudotuberculosis strain at constant temperature, which comprises the primer group capable of amplifying the specific base sequence of the Yersinia pseudotuberculosis genome. In the kit of the present invention, the primer set capable of amplifying the Yersinia pseudotuberculosis genome-specific base sequence includes, but is not limited to, a primer sequence including a part of a nucleic acid sequence at position 768567-768785 bp of a genome (GI No. 153946813) or a part of a complementary strand thereof; the primer includes but is not limited to the primer set a. But not limited to, a primer set having a homology of 50% or more with the aforementioned primer sequence or a single sequence in the complementary strand sequence thereof; including but not limited to primer set B.
In the kit of the present invention, the primer set capable of amplifying the Yersinia pseudotuberculosis genome-specific base sequence may or may not comprise one or more loop primers; the loop primer serves as an optional component. The loop primer is LF and/or LB. The primer set comprising the loop primer LF and/or LB includes, but is not limited to, primer sets A ', B', etc. In a specific embodiment, the kit of the invention may comprise 0.4-1.0. mu. mol/L of LF and/or LB loop primers. In a specific embodiment, the sequences of the primer sets are respectively the primers shown by FIP, BIP, F3, B3, LF and LB, or the primers with 50% or more homology to the single primer of the aforementioned sequence or its complementary strand sequence.
The kit also comprises Bst DNA polymerase buffer solution, Bst DNA polymerase, dNTP solution and Mg2+(MgSO4Or MgCl2) And betaine. In a specific embodiment, the enzyme reaction system of the kit comprises 1 XBst DNA polymerase reaction buffer solution and 2-9mmol/L Mg2+(MgSO4Or MgCl2) 1.0-1.6mmol/LdNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4，0.1％TritonX-100，2mM MgSO4. MgSO in 1 XBst DNA polymerase reaction buffer4And magnesium ion Mg in enzyme reaction system2+And (6) merging.
The kit of the invention also comprises a positive control template. In a specific embodiment, the positive control template includes, but is not limited to, Yersinia pseudotuberculosis whole genomic DNA, partial genomic DNA, or a vector comprising Yersinia pseudotuberculosis whole genomic DNA or partial genomic DNA.
The kit of the invention further comprises a negative control template, and the negative control template comprises but is not limited to double distilled water.
The kit further comprises a color developing agent, wherein the color developing agent comprises but is not limited to calcein, SYBR Green I or hydroxynaphthol blue. When the color developing agent is calcein, the kit also comprises [ Mn2+]For example, MnCl2。
The kit of the invention also comprises double distilled water.
The kit of the invention also comprises a nucleic acid extraction reagent.
The invention also provides a carrier, which comprises any one primer selected from the primer groups A, B, A 'and B'. The vector contains a DNA sequence with the specificity of the Yersinia pseudotuberculosis, so the vector can be applied to the research fields of microbial taxonomy, comparative genomics, evolution and the like, and the application fields of microbial detection and the like. The vector may be, but is not limited to, a plasmid vector (e.g., pBR322, pUC18, pUC19, pBluescript M13, Ti plasmid, etc.), a viral vector (e.g., lambda phage, etc.), and an artificial chromosome vector (e.g., bacterial artificial chromosome BAC, yeast artificial chromosome YAC, etc.). For example, vector pBR322-A containing any one of the primers of primer set A, vector pBR322-B containing any one of the primers of primer set B, and vector pBR322-B 'containing any one of the primers of primer set B' … …. A vector lambda phage-A containing any one of the primers of the primer set A, a vector lambda phage-B containing any one of the primers of the primer set B, … … a vector lambda phage-B 'containing any one of the primers of the primer set B', and the like.
The invention also provides application of the primers selected from any one of the primer groups A, B, A 'and B' in constant-temperature detection of Yersinia pseudotuberculosis.
The invention also provides application of the kit in constant-temperature detection of Yersinia pseudotuberculosis.
The invention also provides application of the vector in constant-temperature detection of Yersinia pseudotuberculosis.
The invention provides a simple, rapid and sensitive method for detecting yersinia pseudotuberculosis, a primer/primer group and a detection reagent/kit for the technical field of food safety detection, and has great significance for food safety in China. The beneficial effects of the invention include: the detection method of the yersinia pseudotuberculosis has the advantages of strong specificity, high sensitivity, short detection time, simple result judgment, convenient operation, low cost and the like. Compared with the current common detection method, the constant temperature amplification method adopted by the invention can be carried out under the constant temperature condition, only a simple constant temperature device is needed, expensive instruments in PCR experiments are not needed, and the steps of carrying out electrophoresis detection on the amplified products and the like are not needed, so the method is very suitable for being widely applied to various social fields including basic food safety detection departments for popularization and use, and can be fully applied even under the environment with relatively insufficient professional knowledge and skill base of molecular biology. Any combination of the above preferred conditions is within the scope of the present invention based on the general knowledge in the art.
Drawings
FIG. 1 shows the specificity of the Yersinia pseudotuberculosis isothermal detection method of example 7 of the present invention.
FIG. 2 shows the sensitivity of the Yersinia pseudotuberculosis detection method of example 8 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
Examples 1-6 Yersinia pseudotuberculosis isothermal reaction System and detection method
The detection is carried out according to the following steps (1) to (3):
(1) extraction of genomic DNA
The Yersinia pseudotuberculosis strain for detection is from China medical bacterial strain preservation management center, and is numbered CMCC 53504. 1mL of the bacterial culture was used to extract genomic DNA and DNA OD using a bacterial nucleic acid extraction kit from Beijing Tiangen bioengineering Co260/OD280At a concentration of 1.8, 18.94 ng/. mu.L.
(2) The method comprises the steps of taking the genomic DNA of the yersinia pseudotuberculosis to be detected as a template, respectively adopting self-matched kits (shown in table 2 and table 3), preparing a reaction system according to the conditions in table 3, and carrying out constant-temperature amplification reaction by taking a specific amplification primer group of the yersinia pseudotuberculosis as a primer. The primers used in examples 1 to 6 were primer set A, A '(1 loop primer), A' (2 loop primer), B, B '(2 loop primer), and B' (1 loop primer), respectively.
(3) The amplification results were confirmed by electrophoresis, turbidity or color development under the conditions shown in Table 3.
As can be seen from Table 3, the detection method and the primer set and reaction system adopted by the detection method can well amplify specific fragments of Yersinia pseudotuberculosis and obtain detection results. In addition, when the detection is performed by using a detector, the detection effect is good when the reaction time is shortened to 10min (as in example 6). Therefore, the present invention can be applied to the detection of the presence or absence of Yersinia pseudotuberculosis in a sample.
Example 7 specific detection of Yersinia pseudotuberculosis
Yersinia pseudotuberculosis 29 strains (1 to 24, 26 to 30 in Table 4 and FIG. 1) were collected, cultured separately from Yersinia pseudotuberculosis strains (25 in Table 4 and FIG. 1), 1mL of the bacterial solution was taken, bacterial DNA was extracted using kit IA, LAMP amplification (primer set A) and visualization by addition of a color-developing agent were carried out separately with reference to the reaction system and conditions of example 1.
The results are shown in Table 4 and FIG. 1, in FIG. 1, 1-24 are Staphylococcus aureus, Staphylococcus aureus subspecies aureoflavus, Staphylococcus epidermidis, Rhodococcus equi, Bacillus cereus, Bacillus mycoides, Listeria monocytogenes, Listeria inowhose, Listeria ehelii, Salmonella enterica subspecies enterica, Salmonella enteritidis, Salmonella typhimurium, Salmonella paratyphi B, Shigella dysenteriae, Shigella boydii, Shigella flexneri, Escherichia coli (containing Clostridium botulinum type A gene), pathogenic Escherichia coli, Escherichia coli diarrheal, enterotoxigenic Escherichia coli, Escherichia coli hemorrhagic, Cronobacter sakazakii and Yersinia enterocolitica, 26-30 are Vibrio vulnificus, Vibrio parahaemolyticus, and Yersinia enterocolitica, respectively, Vibrio freundii, vibrio cholerae and shigella sonnei, NTC: negative control, 25: yersinia pseudotuberculosis. In FIG. 1, the product after the amplification reaction of only Yersinia pseudotuberculosis strain appeared in bright green color as a positive result, as shown in the 25 th tube. The products of other Yersinia pseudotuberculosis strains and the negative control amplification reaction are orange, and are negative results, as shown in tubes No. 1-24, No. 26-30 and NTC negative control tube.
As can be seen from the results shown in FIG. 1 and Table 4, the detection kit and the detection method of the present invention have good Yersinia pseudotuberculosis strain specificity, i.e., only Yersinia pseudotuberculosis strains are amplified positively, and other Yersinia pseudotuberculosis strains are negative.
Preparing a detection kit, wherein the primers adopted in the kit are respectively a primer group B and primer groups A 'and B', and the same detection results are obtained according to the specific detection method, namely, the products of the Yersinia pseudotuberculosis strain and the negative control amplification reaction are negative results, and the products of the Yersinia pseudotuberculosis strain after the amplification reaction are positive results.
In addition, theoretical analysis was performed on the specificity of each primer set a, B, a ', B' according to the method described in table 1, and as a result, it was found that, when at most three mismatches were allowed for each primer, at most five primers were simultaneously aligned to yersinia non-pseudotuberculosis in each primer set, indicating that the specificity of each primer set was better.
Example 8 sensitivity detection
DNA of bacterial CMCC53504 was extracted as in example 1, and subjected to LAMP amplification (primer set A) and visualization by adding a color-developing agent, respectively, under the conditions of the kit IB, 50ng, 5ng, 500pg, 50pg, 5pg, 500fg and 50fg DNA gradient addition reaction system, and other reaction conditions, as in example 1 of Table 3. As shown in fig. 2, 1-7 are 50ng, 5ng, 500pg, 50pg, 5pg, 500fg and 50fg, respectively, ntc: and (5) negative control. In FIG. 2, the reaction products of 50ng, 5ng, and 500pg treatments appeared bright green and as positive results, and the reaction products of 50pg, 5pg, 500fg, and 50fg treatments and the negative control appeared orange and as negative results. The test results showed that each reaction tube contained a minimum of 500pg (about 10 pg equivalent)5Individual bacteria) can still be detected.
According to the detection method, the DNA as low as 5 pg-500 fg in each reaction tube can still be detected by using the primer group B, the primer groups A 'and B' respectively according to the other steps and conditions.
Example 9 commonality testing
Theoretical analysis of the versatility of each of the primer sets A, B and A ', B' was carried out according to the method described in Table 1, and it was found that the primer regions of each of the primer sets perfectly matched with 4 strains of Yersinia pseudotuberculosis (GI Nos. 51594359, 153946813, 170022262 and 186893344, respectively), and could be theoretically used for the detection of the above 4 strains of Yersinia pseudotuberculosis, indicating that the versatility of each of the primer sets was good.
TABLE 1 analysis of the versatility and specificity of primers in the existing detection methods for Yersinia pseudotuberculosis
Figure BDA0001099853460000111
Note: a) the sequence between primers F3 and B3 in the patent is subjected to Bowtie alignment with 4 genomes of Yersinia pseudotuberculosis (GI No. 153946813, 51594359, 170022262 and 186893344 respectively), and the position of a detection region in the GI No. 153946813 genome is determined. b) And performing Blast comparison on the detection region sequences in public database resources, wherein the primer regions are completely matched and have good universality. c) Performing Blast comparison on the detection region sequence in public database resources, wherein the higher the matching degree of the primer region is, the worse the specificity is; if the primers can not be simultaneously compared with the Yersinia pseudotuberculosis strain, the specificity is good.
TABLE 2 types and main components of kit for isothermal detection of Yersinia pseudotuberculosis
Figure BDA0001099853460000112
TABLE 3 examples 1-6 reaction conditions and test results in the method for isothermal detection of Yersinia pseudotuberculosis of the present invention
Figure BDA0001099853460000122
TABLE 4 strains used in the test and the results
Figure BDA0001099853460000132
Figure BDA0001099853460000141
Note: a) CGMCC: china general microbiological culture Collection center, CICC: china center for preservation and management of industrial microbial strains, CMCC: china medical bacteria strain preservation and management center. b) +: positive result, -: and (5) negative result.
Figure IDA0001099853530000011
Figure IDA0001099853530000021
Figure IDA0001099853530000031

Claims (5)

1. A rapid isothermal detection method of yersinia pseudotuberculosis for non-diagnostic purposes, comprising the steps of:
(1) extracting genome DNA from a sample to be detected;
(2) taking the genome DNA as a template, taking a primer group capable of amplifying the specific base sequence of the Yersinia pseudotuberculosis genome as a primer, and carrying out constant-temperature amplification reaction in an enzyme reaction system;
(3) determining whether the sample to be detected has yersinia pseudotuberculosis or not by judging whether the reaction result is positive or not;
wherein the Yersinia pseudotuberculosis genome specific alkali sequence is 768567-768785 bp bit sequence of the Yersinia pseudotuberculosis genome with GI number of 153946813;
wherein the primer group capable of amplifying the specific base sequence of the Yersinia pseudotuberculosis genome is a primer group A or a primer group A';
primer set a:
upstream outer primer F3_ a: 5'-GTTGTTGTATGAATTGCTGG-3' (SEQ ID NO: 1);
downstream outer primer B3_ a: 5'-GCGGATAACTCCTGTTCT-3' (SEQ ID NO: 2);
upstream inner primer FIP _ A: 5'-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3' (SEQ ID NO: 3);
the downstream inner primer BIP _ A: 5'-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3' (SEQ ID NO: 4);
primer set a':
upstream outer primer F3_ a: 5'-GTTGTTGTATGAATTGCTGG-3', respectively;
downstream outer primer B3_ a: 5'-GCGGATAACTCCTGTTCT-3', respectively;
upstream inner primer FIP _ A: 5'-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3', respectively;
the downstream inner primer BIP _ A: 5'-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3', respectively;
upstream loop primer LF _ a: 5'-TATGCTCATCCAGGCGTTTC-3' (SEQ ID NO: 9);
and/or, the downstream loop primer LB _ A: 5'-TGATGGCGATGGGGAAAATT-3' (SEQ ID NO: 10).
2. The method of claim 1, wherein in step (2), the enzymatic reaction system comprises: 1 XBstDNA polymerase reaction buffer, 2-9mmol/L Mg2+1.0-1.6mmol/L dNTP, 0.8-2.0. mu. mol/L FIP _ A and BIP _ A primers, 0.15-0.3. mu. mol/L F3_ A and B3_ A primers, 0.16-0.64U/. mu.L Bst DNA polymerase, 0-1.5mol/L betaine, and 0.4-1.0. mu. mol/L LF _ A and/or LB _ A primers.
3. The method of claim 1, wherein the isothermal amplification reaction is performed by incubating at ① 60-65 ℃ for 10-90 min and terminating at ② 80 ℃ for 2-20 min.
4. The primer used in the method for detecting Yersinia pseudotuberculosis at constant temperature according to claim 1, wherein the primer is a primer group capable of amplifying Yersinia pseudotuberculosis genome-specific base sequence having a part of nucleic acid sequence at positions 768567-768785 bp of Yersinia pseudotuberculosis genome with GI number 153946813 or a part of complementary strand thereof;
wherein the primer group capable of amplifying the specific base sequence of the Yersinia pseudotuberculosis genome is a primer group A or a primer group A';
primer set a:
upstream outer primer F3_ a: 5'-GTTGTTGTATGAATTGCTGG-3', respectively;
downstream outer primer B3_ a: 5'-GCGGATAACTCCTGTTCT-3', respectively;
upstream inner primer FIP _ A: 5'-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3', respectively;
the downstream inner primer BIP _ A: 5'-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3', respectively; primer set a':
upstream outer primer F3_ a: 5'-GTTGTTGTATGAATTGCTGG-3', respectively;
downstream outer primer B3_ a: 5'-GCGGATAACTCCTGTTCT-3', respectively;
upstream inner primer FIP _ A: 5'-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3', respectively;
the downstream inner primer BIP _ A: 5'-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3', respectively;
upstream loop primer LF _ a: 5'-TATGCTCATCCAGGCGTTTC-3', respectively;
and/or, the downstream loop primer LB _ A: 5'-TGATGGCGATGGGGAAAATT-3' are provided.
5. Use of a primer for isothermal detection of yersinia pseudotuberculosis for non-diagnostic purposes, said primer being according to claim 4.
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