CN110938677B - Quick constant-temperature detection method for yersinia pseudotuberculosis nucleic acid and application - Google Patents
Quick constant-temperature detection method for yersinia pseudotuberculosis nucleic acid and application Download PDFInfo
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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
The application is filed on 2016, 8 months and 30 days, has an application number of 201610767506.4 and is named as follows: the divisional application of the Chinese invention patent application of 'a method for rapidly detecting yersinia pseudotuberculosis at constant temperature, a primer and application'; the parent application claims the priority of the Chinese patent application with the application date of 2015, 9 and 2, the application number of 201510556917.4, named as 'method, primer and kit for rapid isothermal detection of cronobacter sakazakii'.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method, a primer 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 Yersinia pseudotuberculosis detection method 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 for the detection process of food-borne pathogenic bacteria in 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, Oyayaama H, Masubuchi H, Yonekawa T, Watanabe K, Nuino N, Hase T. loop-mediated isothermal amplification (E63, J.2000, J.3828, J.3838). The technology can be completed under 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 some organism to be detected into an online website (http:// primer. jp/e) designed by LAMP primer, 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 CN 101182575B and CN 101200760A as examples, the detection of Yersinia pseudotuberculosis is carried out by LAMP technology aiming at the specific sequences of Yersinia pseudotuberculosis, namely 16S-23S region 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 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 invention provides a method for detecting the primer specificity, which is characterized in that the primer specificity is insufficient, and the universality can not be ensured in the prior art as shown in the table 1. 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, convenient 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 comprises the steps of extracting genome DNA from a sample to be detected, carrying out constant-temperature amplification reaction by taking the genome DNA as a template and a Yersinia pseudotuberculosis specific amplification primer group as a primer, and then determining whether 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 base sequence of the Yersinia pseudotuberculosis is 769086-769323 bp bit sequence of Yersinia pseudotuberculosis with GI number of 153946813.
In the present invention, the primer set capable of amplifying the genome-specific nucleotide sequence of Yersinia pseudotuberculosis is a part of the nucleic acid sequence of 769086 to 769323bp of the genome (GI # 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 specific base sequence of the Yersinia pseudotuberculosis genome comprises but not limited to a primer group A, or any one group selected from the primer group with 61 percent or more homology with a single sequence in the sequence of the primer group or the complementary strand sequence thereof.
Primer set a:
the upstream outer primer F3_ A: 5'-GTCGTTACTGGCTGTGGATG-3' (SEQ ID NO: 1);
downstream outer primer B3_ a: 5'-TTGCCAGCGTAAAGTCGG-3' (SEQ ID NO: 2);
upstream inner primer FIP _ A: 5'-TGATTTCTCATTAGACGCCTGCCGTACCGATGGACAACAAACCC-3' (SEQ ID NO: 3);
the downstream inner primer BIP _ A: 5'-GGGGCCACGACCATTATCAAGCAACGCCACTTTCCAAGCG-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 61% 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:
the upstream outer primer F3_ B: 5'-GTCGTTACTGGCTGTGGATG-3' (SEQ ID NO: 5);
downstream outer primer B3_ B: 5'-GCCATCATTGCCAGCGTA-3' (SEQ ID NO: 6) (61% homology to primer B3_ A5'-TTGCCAGCGTAAAGTCGG-3')
An upstream inner primer FIP _ B: 5'-TGATTTCTCATTAGACGCCTGCCGTACCGATGGACAACAAACCC-3' (SEQ ID NO: 7);
the downstream inner primer BIP _ B: 5'-GGGGCCACGACCATTATCAAGCAACGCCACTTTCCAAGCG-3' (SEQ ID NO: 8).
In the method of the present invention, the primer set capable of amplifying the genome-specific base sequence of Yersinia pseudotuberculosis may include, but is not limited to, a loop primer. Preferably, the loop primer is one, and is a loop primer LF. The primer group capable of amplifying the genome specific base sequence of the Yersinia pseudotuberculosis 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 61% or more with a single sequence in the sequences of said primer groups A ', B' or the complementary strand sequences thereof:
a primer set A':
the upstream outer primer F3_ A: 5'-GTCGTTACTGGCTGTGGATG-3', respectively;
downstream outer primer B3_ a: 5'-TTGCCAGCGTAAAGTCGG-3';
upstream inner primer FIP _ a: 5'-TGATTTCTCATTAGACGCCTGCCGTACCGATGGACAACAAACCC-3';
the downstream inner primer BIP _ A: 5'-GGGGCCACGACCATTATCAAGCAACGCCACTTTCCAAGCG-3';
upstream loop primer LF _ a: 5'-TCCAAGAGTCCAACGTGGCAAA-3' (SEQ ID NO: 9);
a primer set B':
the upstream outer primer F3_ B: 5'-GTCGTTACTGGCTGTGGATG-3';
downstream outer primer B3_ B: 5'-GCCATCATTGCCAGCGTA-3', respectively;
an upstream inner primer FIP _ B: 5'-TGATTTCTCATTAGACGCCTGCCGTACCGATGGACAACAAACCC-3';
a downstream inner primer BIP _ B: 5'-GGGGCCACGACCATTATCAAGCAACGCCACTTTCCAAGCG-3';
upstream loop primer LF _ B: 5'-TCCAAGAGTCCAACGTGGCAAA-3' (SEQ ID NO: 10).
In a specific embodiment (including a loop primer), the enzyme reaction system for isothermal amplification is as follows: 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, 0.4-1.0 mu mol/L LF primer, 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 improvement of reaction efficiency. For example, 1 × Bst 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-100,2mM MgSO4.1 XBst DNA polymerase reaction buffer solutionMgSO (2)4And magnesium ion Mg in enzyme reaction system2+And (6) merging.
In the method, the reaction procedure of the isothermal amplification reaction is incubation for 10-90 min at 60-65 ℃, preferably 10-60 min; ② stopping the reaction for 2-20 min at 80 ℃. The invention is not limited to the implementation of the detection method of the invention by other suitable reaction procedures.
In the method of the present invention, the detection method includes, but is not limited to, electrophoresis detection, turbidity detection, or color development detection. The electrophoresis detection is preferably a gel electrophoresis detection method, and can be agarose gel or polyacrylamide gel. In the electrophoresis detection result, if the electrophoresis pattern 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 in Yersinia pseudotuberculosis and contains Yersinia pseudotuberculosis; if no turbidity is found, the sample to be tested is negative to the Yersinia pseudotuberculosis. Or, the bottom of the reaction tube can be visually observed whether the reaction tube has sediment after centrifugation, if the bottom of the reaction tube has sediment, 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 SYBR Green I (30-50 ×), 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; if the color after the reaction is sky blue, the sample to be detected is positive to the Yersinia pseudotuberculosis. The color development detection can detect the reaction result in real time or at the end point through a detection instrument besides observing the reaction result through naked eyes, and through reasonably setting a threshold value of 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, adding 50 μ M calcein into enzyme reaction system, and adding 0.6-1mM [ Mn ]2+]For example, 0.6-1mM of 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 769086-769323 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 is selected from any one of the primer groups with 61 percent or more of single sequence homology 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 61% 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:
the upstream outer primer F3_ A: 5'-GTCGTTACTGGCTGTGGATG-3';
downstream outer primer B3_ a: 5'-TTGCCAGCGTAAAGTCGG-3';
upstream inner primer FIP _ A: 5'-TGATTTCTCATTAGACGCCTGCCGTACCGATGGACAACAAACCC-3', respectively;
the downstream inner primer BIP _ A: 5'-GGGGCCACGACCATTATCAAGCAACGCCACTTTCCAAGCG-3';
primer set B:
upstream outer primer F3_ B: 5'-GTCGTTACTGGCTGTGGATG-3';
downstream outer primer B3_ B: 5'-GCCATCATTGCCAGCGTA-3';
an upstream inner primer FIP _ B: 5'-TGATTTCTCATTAGACGCCTGCCGTACCGATGGACAACAAACCC-3', respectively;
a downstream inner primer BIP _ B: 5'-GGGGCCACGACCATTATCAAGCAACGCCACTTTCCAAGCG-3' is added.
In the primer used in the method for detecting Yersinia pseudotuberculosis at constant temperature, the primer group capable of amplifying the genome-specific base sequence of Yersinia pseudotuberculosis can also comprise but is not limited to a loop primer; preferably, the loop primer is one, and is LF. The primer group capable of amplifying the genome specific base sequence of the Yersinia pseudotuberculosis 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 61% or more with a single sequence in the sequences of said primer groups A ', B' or the complementary strand sequences thereof:
primer set a':
upstream outer primer F3_ a: 5'-GTCGTTACTGGCTGTGGATG-3', respectively;
downstream outer primer B3_ a: 5'-TTGCCAGCGTAAAGTCGG-3', respectively;
upstream inner primer FIP _ A: 5'-TGATTTCTCATTAGACGCCTGCCGTACCGATGGACAACAAACCC-3';
the downstream inner primer BIP _ A: 5'-GGGGCCACGACCATTATCAAGCAACGCCACTTTCCAAGCG-3', respectively;
upstream loop primer LF _ a: 5'-TCCAAGAGTCCAACGTGGCAAA-3', respectively;
a primer set B':
the upstream outer primer F3_ B: 5'-GTCGTTACTGGCTGTGGATG-3', respectively;
downstream outer primer B3_ B: 5'-GCCATCATTGCCAGCGTA-3';
upstream inner primer FIP _ B: 5'-TGATTTCTCATTAGACGCCTGCCGTACCGATGGACAACAAACCC-3';
the downstream inner primer BIP _ B: 5'-GGGGCCACGACCATTATCAAGCAACGCCACTTTCCAAGCG-3';
upstream loop primer LF _ B: 5'-TCCAAGAGTCCAACGTGGCAAA-3' is added.
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 genome specific base sequence of the Yersinia pseudotuberculosis. In the kit of the present invention, the primer set capable of amplifying a Yersinia pseudotuberculosis genome-specific base sequence includes, but is not limited to, a primer sequence including a part of a nucleic acid sequence of 769086 to 769323bp position of genome (GI No. 153946813) or a part of complementary strand thereof; the primer includes but is not limited to the primer set a. But not limited to, a primer group having 61% or more homology with a single sequence in the aforementioned primer sequence or its complementary strand sequence; including but not limited to primer set B.
In the kit of the invention, the primer group capable of amplifying the Yersinia pseudotuberculosis genome specific base sequence may include but is not limited to a loop primer; the loop primer serves as an optional component. Preferably, the loop primer is one and is LF. The primer set including the loop primer LF 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 loop primer. In one embodiment, the sequences of the primer sets are FIP, BIP, F3, B3, and LF, respectively, or primers having 61% or more homology to the above sequences or their complementary strand sequences.
The kit also comprises Bst DNA polymerase buffer solution, Bst DNA polymerase, dNTP solution and Mg2+(MgSO4Or MgCl2) And betaine. In one embodiment, the invention contemplatesThe kit enzyme reaction system comprises 1 XBst DNA polymerase reaction buffer solution and 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. 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-100,2mM MgSO4. MgSO in 1 XBst DNA polymerase reaction buffer4And magnesium ion Mg in enzyme reaction system2+And (6) merging.
The kit of the invention further 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 also 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 vector, which comprises any one group of primers selected from the group consisting of 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 yersinia pseudotuberculosis detection method 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 isothermal amplification method adopted by the invention can be carried out under the isothermal condition, only a simple isothermal device is needed, expensive instruments in PCR experiments are not needed, and steps such as electrophoresis detection and the like on amplification products are not needed, so the method is very suitable for being widely applied to various social fields including basic food safety detection departments to be popularized and used, and can be fully applied even under the environment with relatively insufficient professional knowledge and skill base in 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 Yersinia pseudotuberculosis isothermal detection method in 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 are intended to be included within the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is to be determined by the appended claims. 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 constant temperature 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 sets A, A, A ', B, B', 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 the 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
28 strains of Yersinia pseudotuberculosis (1 to 24, 26 to 29 in Table 4 and FIG. 1) were collected, and cultured separately from Yersinia pseudotuberculosis (25 in Table 4 and FIG. 1), 1mL of the bacterial solution was taken, bacterial DNA was extracted using kit IA, and LAMP amplification (primer set A) and visualization by addition of a color developer were performed, respectively, 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, 26-29 are Vibrio vulnificus, Vibrio parahaemolyticus, and Yersinia enterocolitica, respectively, Vibrio freundii and vibrio cholerae, 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. 26-29 and NTC negative control tube.
As can be seen from the results of 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, while 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 bacterium CMCC53504 was extracted as in example 2, and added to the reaction system in the form of DNA gradient of 500pg, 50pg, 5pg, 500fg and 50fg using kit IIB, and LAMP amplification (primer set A) and visualization by adding color developer were carried out respectively under the other reaction conditions according to the method of example 2 in Table 3. As shown in fig. 2, 1 to 5 are 500pg, 50pg, 5pg, 500fg and 50fg, respectively, NTC: and (5) negative control. In FIG. 2, the reaction products of 500pg, 50pg and 5pg treatments appeared bright green and positive results, and the reaction products of 500fg and 50fg treatments and the negative control appeared orange and negative results. The results of the tests showed that each reaction tube contained a minimum of 5pg (corresponding to about 10 pg)3Individual 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, in the same other steps and conditions as above.
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
Note: a) the sequence between primers F3 and B3 in the patent is subjected to Bowtie alignment with 4 genomes of Yersinia pseudotuberculosis (GI Nos. 153946813, 51594359, 170022262 and 186893344 respectively), and the position of the 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 sequences in public database resources, wherein the higher the matching degree of the primer regions is, the worse the specificity is; if the primers can not be simultaneously compared on the Yersinia pseudotuberculosis strain, the specificity is good.
TABLE 2 kinds and main components of kit for constant temperature detection of Yersinia pseudotuberculosis
TABLE 3 examples 1 to 6 reaction conditions and results of detection in the method for isothermal detection of Yersinia pseudotuberculosis of the present invention
TABLE 4 strains used in the test and the results
Note: a) CGMCC: china center for preservation of common microbial strains, 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.
<110> Shanghai Wangwang food group Co., Ltd., Shanghai Marine industry and technology research institute
Rapid constant-temperature detection method for yersinia pseudotuberculosis nucleic acid and application
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Claims (9)
1. A rapid isothermal detection method of yersinia pseudotuberculosis for non-diagnostic purposes, comprising the following steps:
(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 base sequence is 769086-769323 bp bit sequence of Yersinia pseudotuberculosis genome with GI number of 153946813;
the primer group capable of amplifying the specific base sequence of the Yersinia pseudotuberculosis genome is a primer group B or a primer group B';
primer set B:
upstream outer primer F3_ B: 5'-GTCGTTACTGGCTGTGGATG-3' (SEQ ID NO: 5);
downstream outer primer B3_ B: 5'-GCCATCATTGCCAGCGTA-3' (SEQ ID NO: 6);
upstream inner primer FIP _ B: 5'-TGATTTCTCATTAGACGCCTGCCGTACCGATGGACAACAAACCC-3' (SEQ ID NO: 7);
the downstream inner primer BIP _ B: 5'-GGGGCCACGACCATTATCAAGCAACGCCACTTTCCAAGCG-3' (SEQ ID NO: 8);
a primer set B':
the upstream outer primer F3_ B: 5'-GTCGTTACTGGCTGTGGATG-3', respectively;
downstream outer primer B3_ B: 5'-GCCATCATTGCCAGCGTA-3', respectively;
upstream inner primer FIP _ B: 5'-TGATTTCTCATTAGACGCCTGCCGTACCGATGGACAACAAACCC-3', respectively;
the downstream inner primer BIP _ B: 5'-GGGGCCACGACCATTATCAAGCAACGCCACTTTCCAAGCG-3', respectively;
upstream loop primer LF _ B: 5'-TCCAAGAGTCCAACGTGGCAAA-3' (SEQ ID NO: 10).
2. The method of claim 1, wherein in step (2), the enzymatic reaction system comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg2+1.0-1.6mmol/L dNTP, 0.8-2.0 μmol/L FIP _ B and BIP _ B primers, 0.15-0.3 μmol/L F3_ B and B3_ B primers, 0.16-0.64U/μ L Bst DNA polymerase, 0-1.5mol/L betaine, including or not including 0.4-1.0 μmol/L LF _ B.
3. The method of claim 1, wherein the isothermal amplification reaction is performed by a reaction sequence comprising: incubating for 10-90 min at 60-65 ℃; ② stopping the reaction for 2-20 min at 80 ℃.
4. The primer used in the method for isothermal detection of Yersinia pseudotuberculosis of claim 1, wherein the primer is a primer set capable of amplifying Yersinia pseudotuberculosis genome-specific base sequence that is a part of a nucleic acid sequence at positions 769086-769323 bp of Yersinia pseudotuberculosis genome with GI number 153946813 or a part of a complementary strand thereof;
the primer group capable of amplifying the specific base sequence of the Yersinia pseudotuberculosis genome is a primer group B or a primer group B';
primer set B:
upstream outer primer F3_ B: 5'-GTCGTTACTGGCTGTGGATG-3' (SEQ ID NO: 5);
downstream outer primer B3_ B: 5'-GCCATCATTGCCAGCGTA-3' (SEQ ID NO: 6);
an upstream inner primer FIP _ B: 5'-TGATTTCTCATTAGACGCCTGCCGTACCGATGGACAACAAACCC-3' (SEQ ID NO: 7);
a downstream inner primer BIP _ B: 5'-GGGGCCACGACCATTATCAAGCAACGCCACTTTCCAAGCG-3' (SEQ ID NO: 8);
a primer set B':
upstream outer primer F3_ B: 5'-GTCGTTACTGGCTGTGGATG-3', respectively;
downstream outer primer B3_ B: 5'-GCCATCATTGCCAGCGTA-3', respectively;
upstream inner primer FIP _ B: 5'-TGATTTCTCATTAGACGCCTGCCGTACCGATGGACAACAAACCC-3';
the downstream inner primer BIP _ B: 5'-GGGGCCACGACCATTATCAAGCAACGCCACTTTCCAAGCG-3', respectively;
upstream loop primer LF _ B: 5'-TCCAAGAGTCCAACGTGGCAAA-3' (SEQ ID NO: 10).
5. A kit for isothermal detection of Yersinia pseudotuberculosis, comprising the primer of claim 4.
6. The kit of claim 5, further comprising a Bst DNA polymerase reaction buffer, Bst DNA polymerase, dNTP solution, Mg2+And one or more of betaine.
7. The kit of claim 5, wherein the enzymatic reaction system of the kit comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg2+1.0-1.6mmol/L dNTP, 0.8-2.0 μmol/L FIP _ B and BIP _ B primers, 0.15-0.3 μmol/L F3_ B and B3_ B primers, 0.16-0.64U/μ L Bst DNA polymerase, 0-1.5mol/L betaine, including or not including 0.4-1.0 μmol/L LF _ B primer.
8. Use of a primer for isothermal detection of yersinia pseudotuberculosis for non-diagnostic purposes, said primer being according to claim 4.
9. Use of a kit according to any one of claims 5 to 7 for isothermal detection of yersinia pseudotuberculosis for non-diagnostic purposes.
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