CN110951837B - Rapid constant-temperature detection method for Listeria monocytogenes nucleic acid and application - Google Patents
Rapid constant-temperature detection method for Listeria monocytogenes 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 Listeria monocytogenes at constant temperature. The method comprises the following steps: extracting genome DNA from a sample to be detected; performing constant-temperature amplification reaction in an enzyme reaction system by using the genome DNA as a template and a primer group capable of amplifying a specific sequence of the Listeria monocytogenes as a primer; and determining whether the Listeria monocytogenes exists in the sample to be detected 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, 30, and has the application number of 201610767436.2 and the name of the invention: the divisional application of the Chinese patent application of 'a method for rapidly detecting Listeria monocytogenes at constant temperature, a primer and application thereof'; 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, primers and a kit for rapidly detecting listeria monocytogenes at a constant temperature.
Background
Listeria monocytogenes (Listeria monocytogenes) is a gram-positive brevibacterium widely found in nature, and may be found in meat, eggs, poultry, seafood, dairy products, and vegetables. It can cause listeriosis in humans and animals, is mainly manifested by septicemia, meningitis and mononucleosis after infection, and is especially harmful to pregnant women, newborns, the elderly and patients with immunodeficiency. In 1986, the World Health Organization (WHO) listed it as one of four general pathogens in food in the 90's of the 20 th century; in 2000, WHO listed it as one of the food-borne pathogens that needed intensive monitoring.
At present, the detection of the listeria monocytogenes at home and abroad is mainly a culture method and a serological detection method, which comprise enrichment, selective culture and biochemical identification. These conventional detection methods have the disadvantages of high labor intensity, time consumption, high cost, and the like. More importantly, the traditional method has low detection sensitivity and insufficient specificity, and particularly, the traditional method is difficult to distinguish strains with similar sources. Therefore, in order to ensure food safety, a rapid, simple and accurate method for detecting listeria monocytogenes 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 (8512, 2000, 63). 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 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. The invention discloses a method for detecting listeria monocytogenes, which takes patents ZL201410632370.7 and ZL201310353402.5 as examples, and respectively aims at specific genes-hlyA gene and prfA gene-of the listeria monocytogenes reported in the literature, and adopts LAMP technology to detect the listeria monocytogenes. 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 versatility and/or specificity in practical applications. The invention provides a method for detecting the primer specificity, which is characterized in that the primer universality is not enough and the specificity can not be ensured in the prior art as shown in Table 1. That is, the listeria monocytogenes detection sequence used in the prior art methods is not actually unique to listeria monocytogenes, i.e., it is possible that listeria monocytogenes other than listeria monocytogenes is erroneously identified as listeria monocytogenes; meanwhile, the listeria monocytogenes detection sequences used in the prior art methods are not common to listeria monocytogenes, i.e., some strains in listeria monocytogenes may be missed. Therefore, a method for detecting listeria monocytogenes capable of ensuring specificity and universality simultaneously 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 primer universality and specificity deficiency in the primer design of the existing 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 Listeria monocytogenes, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention designs a Listeria monocytogenes LAMP primer based on microbial genome data resources (data of 8 months and 5 days as of 2013) in a GenBank database, and provides a method, a primer group and a kit for rapid isothermal amplification detection of Listeria monocytogenes. The detection method for detecting the listeria monocytogenes 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 a Listeria monocytogenes strain, which comprises the following steps:
(1) extracting genome DNA from a sample to be detected;
(2) performing constant-temperature amplification reaction in an enzyme reaction system by using the genome DNA as a template and a primer group capable of amplifying the specific base sequence of the genome of the Listeria monocytogenes as a primer;
(3) and determining whether the Listeria monocytogenes exists in the sample to be detected by judging whether the reaction result is positive or not.
The method for detecting the listeria monocytogenes 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 specific amplification primer group of the listeria monocytogenes as primers, and then determining whether the listeria monocytogenes strain 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 a DNA polymerase reaction system.
In the invention, the genome-specific base sequence of the listeria monocytogenes is the bit sequence of 759029-759351 bp of the listeria monocytogenes with the GI number of 16802048.
In the present invention, the primer set capable of amplifying a genome-specific nucleotide sequence of Listeria monocytogenes is a part of a nucleic acid sequence of 759029 to 759351bp of the genome (GI No. 16802048) or a part of a complementary strand thereof. Wherein the Listeria monocytogenes genome-specific base sequence is a base sequence that is unique to the Listeria monocytogenes genome and is not included in the genome of other microorganisms.
Wherein, the primer group capable of amplifying the specific base sequence of the Listeria monocytogenes genome comprises but is not limited to primer group A, or any one of the primer groups with 61% or more homology with the single sequence in the primer group sequence or the complementary strand sequence thereof.
Primer set a:
upstream outer primer F3_ a: 5'-ACGTAGATTTACTCCGGG-3' (SEQ ID NO: 1);
downstream outer primer B3_ a: 5'-TCTGTTTGATACGTACCTGT-3' (SEQ ID NO: 2);
upstream inner primer FIP _ A: 5'-GCTCGAATGTTGGTGGAACTTCAAGCCCTATGATATTGGTG-3' (SEQ ID NO: 3);
the downstream inner primer BIP _ A: 5'-TCTTAGGCCATCCGTGGTATTCTAAGCCAGTCATCTCAC-3' (SEQ ID NO: 4).
In the present invention, the primer set capable of amplifying the genome-specific base sequence of listeria monocytogenes may further include a primer set having a homology of 61% or more with a single sequence in the sequence of each of the aforementioned primer sets or the sequence of the complementary strand thereof, and the primer set includes, but is not limited to, a primer set B:
primer set B:
the upstream outer primer F3_ B: 5'-TTTACTCCGGGACCTATG-3' (SEQ ID NO: 5) (61% homology to primer F3_ A5'-ACGTAGATTTACTCCGGG-3');
downstream outer primer B3_ B: 5'-TGTTTGATACGTACCTGTTG-3' (SEQ ID NO: 6);
upstream inner primer FIP _ B: 5'-GCTCGAATGTTGGTGGAACTTCAAGCCCTATGATATTGGTG-3' (SEQ ID NO: 7);
the downstream inner primer BIP _ B: 5'-GTCATTTTCAAGAAGCGGAAGAGTAAGCCAGTCATCTCACC-3' (SEQ ID NO: 8).
In the method of the present invention, the primer set capable of amplifying a genome-specific base sequence of listeria monocytogenes 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 genome-specific base sequence of Listeria monocytogenes 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'-ACGTAGATTTACTCCGGG-3', respectively;
downstream outer primer B3_ a: 5'-TCTGTTTGATACGTACCTGT-3', respectively;
upstream inner primer FIP _ A: 5'-GCTCGAATGTTGGTGGAACTTCAAGCCCTATGATATTGGTG-3', respectively;
the downstream inner primer BIP _ A: 5'-TCTTAGGCCATCCGTGGTATTCTAAGCCAGTCATCTCAC-3', respectively;
upstream loop primer LF _ a: 5'-TCAATAACCGGGACAGATGTG-3' (SEQ ID NO: 9);
a primer set B':
upstream outer primer F3_ B: 5'-TTTACTCCGGGACCTATG-3', respectively;
downstream outer primer B3_ B: 5'-TGTTTGATACGTACCTGTTG-3';
an upstream inner primer FIP _ B: 5'-GCTCGAATGTTGGTGGAACTTCAAGCCCTATGATATTGGTG-3', respectively;
a downstream inner primer BIP _ B: 5'-GTCATTTTCAAGAAGCGGAAGAGTAAGCCAGTCATCTCACC-3', respectively;
upstream loop primer LF _ B: 5'-TCAATAACCGGGACAGATGTG-3' (SEQ ID NO: 10);
and/or, the downstream loop primer LB _ B: 5'-TCTTAGGCCATCCGTGGTAT-3' (SEQ ID NO: 11).
In specific embodiments, for example, the primer set B' may include 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 XBst DNA polymerase reaction buffer solution, 2-9mmol/L Mg 2+ (MgSO 4 Or MgCl 2 ) 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 Mg 2+ (MgSO 4 Or MgCl 2 ) 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 reactionThe washing solution can be 1 × Thermopol reaction buffer solution containing 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl, 10mmol/L (NH4) 2 SO4，0.1％Triton X-100，2mM MgSO 4 . MgSO in 1 XBst DNA polymerase reaction buffer 4 And magnesium ion Mg in enzyme reaction system 2+ And (6) merging.
In the method, the reaction procedure of the constant-temperature amplification reaction is incubation at 60-65 ℃ for 10-90 min, preferably 10-60 min; ② terminating 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, 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 image shows a characteristic step-shaped strip, the sample to be detected is positive to the listeria monocytogenes and contains the listeria monocytogenes; if the electrophoretogram does not present a characteristic step-shaped strip, the sample to be detected is negative to the Listeria monocytogenes. 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 listeria monocytogenes and contains the listeria monocytogenes; if no turbidity is found, the sample to be detected is negative to the Listeria monocytogenes. Or whether the reaction tube bottom is precipitated or not can be visually observed after centrifugation, if the reaction tube bottom is precipitated, the sample to be detected is positive to the listeria monocytogenes and contains the listeria monocytogenes; if no sediment is left at the bottom of the reaction tube, the sample to be detected is negative to the Listeria monocytogenes.
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 after reaction is orange, the sample to be detected is negative to the Listeria monocytogenes; if the color after the reaction is green, the sample to be detected is positive to the listeria monocytogenes and contains the listeria monocytogenes. 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 the listeria monocytogenes; if the color after the reaction is sky blue, the sample to be detected is positive for the Listeria monocytogenes. The color development detection can be performed by a detection instrument in real time or end point detection reaction results except for the reaction results observed by naked eyes, and by 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 listeria monocytogenes; and when the reaction result of the sample to be detected is greater than the threshold value, the sample to be detected is positive for the Listeria monocytogenes. 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 the 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 added 2+ ]For example, 0.6-1mM MnCl 2 。
The invention also provides primers for use in a method for isothermal detection of listeria monocytogenes strains. The primer comprises a primer group capable of amplifying a specific base sequence of a Listeria monocytogenes genome, and includes, but is not limited to, a part of a nucleic acid sequence of 759029-759351 bp of the Listeria monocytogenes genome with GI number 16802048 or a part of a complementary strand thereof.
Wherein the primer group capable of amplifying the genome-specific nucleotide sequence of Listeria monocytogenes is selected from any one of the following primer groups, or is selected from any one of the primer groups having 61% or more homology with a single sequence in the sequence of each primer group or the complementary strand sequence thereof. Wherein, the primer group includes but is not limited to primer group A. The primer group having 61% or more homology with a single sequence in the aforementioned primer group sequence or its complementary strand sequence includes, but is not limited to, primer group B.
Primer set a:
upstream outer primer F3_ a: 5'-ACGTAGATTTACTCCGGG-3', respectively;
downstream outer primer B3_ a: 5'-TCTGTTTGATACGTACCTGT-3', respectively;
upstream inner primer FIP _ A: 5'-GCTCGAATGTTGGTGGAACTTCAAGCCCTATGATATTGGTG-3', respectively;
the downstream inner primer BIP _ A: 5'-TCTTAGGCCATCCGTGGTATTCTAAGCCAGTCATCTCAC-3', respectively;
primer set B:
the upstream outer primer F3_ B: 5'-TTTACTCCGGGACCTATG-3', respectively;
downstream outer primer B3_ B: 5'-TGTTTGATACGTACCTGTTG-3', respectively;
upstream inner primer FIP _ B: 5'-GCTCGAATGTTGGTGGAACTTCAAGCCCTATGATATTGGTG-3', respectively;
a downstream inner primer BIP _ B: 5'-GTCATTTTCAAGAAGCGGAAGAGTAAGCCAGTCATCTCACC-3' are provided.
In the primers used in the method for isothermal detection of listeria monocytogenes, the primer set capable of amplifying the genome-specific base sequence of listeria monocytogenes 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 genome-specific base sequence of Listeria monocytogenes 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':
upstream outer primer F3_ a: 5'-ACGTAGATTTACTCCGGG-3', respectively;
downstream outer primer B3_ a: 5'-TCTGTTTGATACGTACCTGT-3', respectively;
upstream inner primer FIP _ A: 5'-GCTCGAATGTTGGTGGAACTTCAAGCCCTATGATATTGGTG-3', respectively;
the downstream inner primer BIP _ A: 5'-TCTTAGGCCATCCGTGGTATTCTAAGCCAGTCATCTCAC-3', respectively;
upstream loop primer LF _ a: 5'-TCAATAACCGGGACAGATGTG-3', respectively;
a primer set B':
upstream outer primer F3_ B: 5'-TTTACTCCGGGACCTATG-3', respectively;
downstream outer primer B3_ B: 5'-TGTTTGATACGTACCTGTTG-3', respectively;
upstream inner primer FIP _ B: 5'-GCTCGAATGTTGGTGGAACTTCAAGCCCTATGATATTGGTG-3', respectively;
the downstream inner primer BIP _ B: 5'-GTCATTTTCAAGAAGCGGAAGAGTAAGCCAGTCATCTCACC-3', respectively;
upstream loop primer LF _ B: 5'-TCAATAACCGGGACAGATGTG-3', respectively;
and/or, the downstream loop primer LB _ B: 5'-TCTTAGGCCATCCGTGGTAT-3' are provided.
In a specific embodiment, the primer set B' may include 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 61% or more homology with single primer in the aforementioned primer sequence or its complementary strand sequence.
The invention also provides a kit used in the method for detecting the listeria monocytogenes strain at constant temperature, which comprises the primer group capable of amplifying the genome-specific base sequence of the listeria monocytogenes strain. In the kit of the present invention, the primer set capable of amplifying a genome-specific nucleotide sequence of Listeria monocytogenes includes, but is not limited to, a portion of a nucleic acid sequence of 759029-759351 bp of the genome (GI: 16802048) or a portion of the complementary strand thereof as the primer sequence; 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 present invention, the primer set capable of amplifying a genome-specific base sequence of listeria monocytogenes 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 61% or more homology with the sequences or the single primer of the complementary strand sequence.
The kit also comprises Bst DNA polymerase buffer solution, Bst DNA polymerase, dNTP solution and Mg 2+ (MgSO 4 Or MgCl 2 ) 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 Mg 2+ (MgSO 4 Or MgCl 2 ) 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 (pH 8.8), 10mmol/L KCl, 10mmol/L (NH4) 2 SO4，0.1％Triton X-100，2mM MgSO 4 . MgSO in 1 XBst DNA polymerase reaction buffer 4 And magnesium ion Mg in enzyme reaction system 2+ 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, a whole genomic DNA, a partial genomic DNA of listeria monocytogenes, or a vector comprising a whole genomic DNA or a partial genomic DNA of listeria monocytogenes.
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 of the invention further comprises a color-developing agent, wherein the color-developing agent comprises but is not limited toIn calcein, SYBR Green I or hydroxynaphthol blue. When the color developing agent is calcein, the kit also comprises [ Mn 2+ ]For example, MnCl 2 。
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 Listeria monocytogenes, so the vector can be applied to the research fields of microbial taxonomy, comparative genomics, evolution and the like, and the application field 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 Listeria monocytogenes.
The invention also provides application of the kit in constant-temperature detection of Listeria monocytogenes.
The invention also provides application of the vector in constant-temperature detection of Listeria monocytogenes.
The invention provides a simple, rapid and sensitive method for detecting Listeria monocytogenes, 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 method for detecting the listeria monocytogenes 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 isothermal detection method for Listeria monocytogenes of example 7 of the present invention.
FIG. 2 shows the sensitivity of the Listeria monocytogenes detection method of embodiment 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, but 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.
Example 1-6 Listeria monocytogenes 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 listeria monocytogenes strain for detection is from the China center for Industrial culture Collection of microorganisms with the number CICC21635 (ATCC 19115). Extracting 1mL of bacterial culture with bacterial nucleic acid extraction kit of Beijing Tiangen bioengineering companyTaking genomic DNA, DNA OD 260 /OD 280 At a concentration of 1.8, 178.8 ng/. mu.L.
(2) Taking the genome DNA of the listeria monocytogenes to be detected as a template, respectively adopting self-prepared kits (shown in tables 2 and 3), preparing a reaction system according to the conditions in the table 3, and taking the specific amplification primer group of the listeria monocytogenes as a primer to carry out constant-temperature amplification reaction. The primers used in examples 1 to 6 were primer set A, A ', B, B' (2-loop primer), B '(1-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 the reaction system adopted by the detection method can well amplify the specific fragment of Listeria monocytogenes and obtain the detection result. 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 invention can be applied to detecting whether the sample contains the listeria monocytogenes.
Example 7 Listeria monocytogenes specific detection
28 strains of listeria monocytogenes (1-6, 8-29 in table 4 and fig. 1) were collected, and these strains and the listeria monocytogenes strain (7 in table 4 and fig. 1) were cultured, respectively, 1mL of the bacterial solution was taken, bacterial DNA was extracted using the kit IA, and LAMP amplification (primer set a) and visualization with color reagent were performed, respectively, with reference to the reaction system and conditions of example 1.
The detection results are shown in Table 4 and FIG. 1, in FIG. 1, 1 to 6 are respectively Staphylococcus aureus, Staphylococcus aureus Chryseozoinum species, Staphylococcus epidermidis, Rhodococcus equi, Bacillus cereus, Bacillus mycoides, 8 to 29 are respectively Listeria innock, Listeria eheli, Salmonella 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 enterohemorrhagic Escherichia coli, Cronobacter sakazakii, Yersinia enterocolitica, Yersinia pseudotuberculosis, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio and Vibrio cholera, NTC: negative control, 7: listeria monocytogenes. In FIG. 1, the product obtained after the amplification reaction of only the Listeria monocytogenes strain appeared bright green and was a positive result, as shown in tube 7. The products of the other non-Listeria monocytogenes strains and the negative control amplification reaction are orange, which is a negative result, as shown in tubes No. 1-6, No. 8-29 and NTC negative control tubes.
As can be seen from the results of FIG. 1 and Table 4, the detection kit and the detection method of the invention have good specificity of Listeria monocytogenes strains, that is, only Listeria monocytogenes strains are amplified positively, and other Listeria monocytogenes strains are not amplified positively.
Preparing a detection kit, wherein the primers adopted in the kit are respectively a primer group B, and primer groups A 'and B' to respectively obtain the same detection results according to the specific detection method, namely, the products after the amplification reaction of the non-monocytic cell proliferation listeria strain and the negative control are negative results, and the products after the amplification reaction of the monocytic cell proliferation listeria strain are positive results.
In addition, theoretical analysis is carried out on the specificity of the primer group A, the primer group B and the primer groups A 'and B' respectively according to the method described in the table 1, and the result shows that under the condition that at most three mismatches are allowed for each primer, at most one primer in each primer group is compared with the Listeria monocytogenes, which indicates that the specificity of each primer group is better.
EXAMPLE 8 sensitivity detection
DNA of the bacterium CICC21635 was extracted by the method of example 1, and LAMP amplification (primer set A) and visualization by adding a color-developing agent were carried out by using kit IB and according to the method of example 1 of Table 3, using DNA gradient of 50ng, 5ng, 500pg, 50pg, 5pg, 500fg and 50fg added to the reaction system. As shown in fig. 2, 1 to 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, 500pg, 50pg, 5pg, and 500fg treatments appeared bright green and as positive results, and the reaction products of 50fg treatments and the negative control appeared orange and as negative results. The detection results show that DNA of 500fg (equivalent to about 100 bacteria) can be detected in each reaction tube, and the sensitivity is high.
According to the detection method, other steps and conditions are the same, the DNA as low as 5 pg-500 fg in each reaction tube can still be detected by using the primer group B and the primer groups A 'and B', and the detection sensitivity is higher.
Example 9 commonality testing
According to the method described in table 1, theoretical analysis was performed on the universality of the primer set A, B and the primer sets a 'and B', respectively, and as a result, it was found that the primer regions of the primer sets and four strains (GI numbers 386025288, 217963303, 386006713, and 404406483, respectively) in the database had a large number of mismatches (> 7) in the primer regions, and completely matched the sequences of the remaining 33 strains of listeria monocytogenes, indicating that the universality of each primer set was good.
TABLE 1 analysis of primer commonality and specificity in existing detection methods for Listeria monocytogenes
Note: a) the sequence between the primers F3 and B3 in the patent is subjected to Bowtie alignment with 37 genomes of the Listeria monocytogenes, and the position of the detection region in the GI No. 16802048 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; the specificity is good when the strain can not be compared with the non-monocyte hyperplasia Listeria monocytogenes simultaneously.
TABLE 2 constant temperature detection of Listeria monocytogenes kit species and major components
TABLE 3 examples 1-6 reaction conditions and test results in the method for isothermal detection of Listeria monocytogenes of the present invention
TABLE 4 strains used in the test and test results
Note: a) CGMCC: china general microbiological culture Collection center, CICC: china center for preservation and management of industrial microbial strains, CMCC: china center for preservation and management of bacterial strains. 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 <120> listeria monocytogenes nucleic acid and application
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Claims (9)
1. A non-diagnostic purpose method for rapid isothermal detection of Listeria monocytogenes, comprising the steps of:
(1) extracting genome DNA from a sample to be detected;
(2) performing constant-temperature amplification reaction in an enzyme reaction system by using the genome DNA as a template and a primer group capable of amplifying the specific base sequence of the genome of the Listeria monocytogenes as a primer;
(3) determining whether the Listeria monocytogenes exists in the sample to be detected by judging whether the reaction result is positive or not;
wherein the genome-specific base sequence of the Listeria monocytogenes is the sequence of 759029-759351 bp bits of the Listeria monocytogenes genome with the GI number of 16802048;
the primer group capable of amplifying the specific base sequence of the genome of the listeria monocytogenes is a primer group B or a primer group B';
primer set B:
upstream outer primer F3_ B: 5'-TTTACTCCGGGACCTATG-3' (SEQ ID NO: 5);
downstream outer primer B3_ B: 5'-TGTTTGATACGTACCTGTTG-3' (SEQ ID NO: 6);
upstream inner primer FIP _ B: 5'-GCTCGAATGTTGGTGGAACTTCAAGCCCTATGATATTGGTG-3' (SEQ ID NO: 7);
the downstream inner primer BIP _ B: 5'-GTCATTTTCAAGAAGCGGAAGAGTAAGCCAGTCATCTCACC-3' (SEQ ID NO: 8);
a primer set B':
the upstream outer primer F3_ B: 5'-TTTACTCCGGGACCTATG-3', respectively;
downstream outer primer B3_ B: 5'-TGTTTGATACGTACCTGTTG-3', respectively;
upstream inner primer FIP _ B: 5'-GCTCGAATGTTGGTGGAACTTCAAGCCCTATGATATTGGTG-3', respectively;
the downstream inner primer BIP _ B: 5'-GTCATTTTCAAGAAGCGGAAGAGTAAGCCAGTCATCTCACC-3', respectively;
upstream loop primer LF _ B: 5'-TCAATAACCGGGACAGATGTG-3' (SEQ ID NO: 10);
and/or, the downstream loop primer LB _ B: 5'-TCTTAGGCCATCCGTGGTAT-3' (SEQ ID NO: 11).
2. The method of claim 1, wherein in step (2), the enzymatic reaction system comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg 2+ 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, and including or not including 0.4-1.0 μmol/L LF _ B and/or LB _ B primers.
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 terminating the reaction for 2-20 min at 80 ℃.
4. The primer used in the method for isothermal detection of listeria monocytogenes according to claim 1, wherein the primer comprises a primer set capable of amplifying a genome-specific base sequence of listeria monocytogenes, wherein the genome-specific base sequence of listeria monocytogenes is a part of a nucleic acid sequence at the 759029-759351 bp position of the genome of listeria monocytogenes with the GI number of 16802048 or a part of a complementary strand thereof;
the primer group capable of amplifying the specific base sequence of the genome of the listeria monocytogenes is a primer group B or a primer group B';
primer set B:
the upstream outer primer F3_ B: 5'-TTTACTCCGGGACCTATG-3' (SEQ ID NO: 5);
downstream outer primer B3_ B: 5'-TGTTTGATACGTACCTGTTG-3' (SEQ ID NO: 6);
upstream inner primer FIP _ B: 5'-GCTCGAATGTTGGTGGAACTTCAAGCCCTATGATATTGGTG-3' (SEQ ID NO: 7);
a downstream inner primer BIP _ B: 5'-GTCATTTTCAAGAAGCGGAAGAGTAAGCCAGTCATCTCACC-3' (SEQ ID NO: 8);
a primer set B':
upstream outer primer F3_ B: 5'-TTTACTCCGGGACCTATG-3', respectively;
downstream outer primer B3_ B: 5'-TGTTTGATACGTACCTGTTG-3', respectively;
upstream inner primer FIP _ B: 5'-GCTCGAATGTTGGTGGAACTTCAAGCCCTATGATATTGGTG-3', respectively;
the downstream inner primer BIP _ B: 5'-GTCATTTTCAAGAAGCGGAAGAGTAAGCCAGTCATCTCACC-3', respectively;
upstream loop primer LF _ B: 5'-TCAATAACCGGGACAGATGTG-3' (SEQ ID NO: 10);
and/or, the downstream loop primer LB _ B: 5'-TCTTAGGCCATCCGTGGTAT-3' (SEQ ID NO: 11).
5. A kit for isothermal detection of Listeria monocytogenes 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, Mg 2+ 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 Mg 2+ 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, and including or not including 0.4-1.0 μmol/L LF _ B and/or LB _ B primers.
8. Use of a primer for isothermal detection of listeria monocytogenes for non-diagnostic purposes, wherein the primer is the primer of claim 4.
9. Use of a kit according to any one of claims 5 to 7 for isothermal detection of listeria monocytogenes for non-diagnostic purposes.
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