CN106367493B - Method, primer and application for rapid constant-temperature detection of salmonella - Google Patents
Method, primer and application for rapid constant-temperature detection of salmonella Download PDFInfo
- Publication number
- CN106367493B CN106367493B CN201610767608.6A CN201610767608A CN106367493B CN 106367493 B CN106367493 B CN 106367493B CN 201610767608 A CN201610767608 A CN 201610767608A CN 106367493 B CN106367493 B CN 106367493B
- Authority
- CN
- China
- Prior art keywords
- primer
- salmonella
- upstream
- genome
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a method, a primer group and a kit for rapidly detecting salmonella 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 the specific sequence of the salmonella as a primer; and determining whether the salmonella exists in the sample to be detected by judging whether the reaction result is positive. 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
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method, primers and a kit for rapidly detecting salmonella at constant temperature.
Background
Salmonella (Salmonella spp.) is a gram-negative bacillus, which inhabits the intestinal tracts of humans and animals, is excreted from the body by feces, and is further transmitted through contaminated water and food, causing symptoms such as gastroenteritis, typhoid and paratyphoid in humans. In recent years, the harm caused by salmonella in the global scope is on the rising trend, and the salmonella is an important pathogenic bacterium in the field of public health. Therefore, the method has important significance for the detection and prevention of the bacteria.
The traditional salmonella 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 throughput, high sensitivity, high specificity, rapidness and convenience in the detection process of food-borne pathogenic bacteria in modern society. In recent years, with the development of nucleic acid molecule detection technology, researchers have developed detection means of PCR and fluorescence PCR technologies, but both methods require special detection instruments, and are not suitable for real-time field detection in basic detection departments, especially in enterprise production lines. In order to ensure the safety of food, a rapid, simple and accurate method for detecting salmonella 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. The invention takes patents ZL201110026963.5 and ZL201110433262.3 as examples, and the patents respectively aim at specific genes of salmonella, i.e. fimY gene and invA gene, reported in literatures, and the LAMP technology is adopted for salmonella detection. 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. Table 1 of the present invention shows the problem that the versatility is not ensured in the prior art. That is, the salmonella detection sequences used in the prior art methods are not actually common to salmonella, i.e., there is a possibility that a partial strain of salmonella may be missed. A similar problem exists in the confirmation of specificity, i.e., the possibility of incorrectly identifying non-salmonella as salmonella. Therefore, a salmonella 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 specific recognition of salmonella, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention provides a method, a primer group and a kit for rapid isothermal amplification detection of salmonella by designing salmonella LAMP primers based on microbial genome data resources (data up to 2013, 8, 5 and 2013) in a GenBank database. The detection method for detecting the salmonella 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 salmonella 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 salmonella genome as a primer;
(3) and determining whether the salmonella exists in the sample to be detected by judging whether the reaction result is positive.
The method for detecting the salmonella strain at constant temperature comprises the steps of extracting genome DNA from a sample to be detected, carrying out constant-temperature amplification reaction by using the genome DNA as a template and a salmonella specific amplification primer group as a primer, and then determining whether salmonella 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 salmonella genome specific alkali sequence is a bit sequence of 4188946-4189180 bp of salmonella with GI number of 194447306.
In the invention, the primer group capable of amplifying the specific base sequence of the salmonella genome is a part of the nucleic acid sequence of 4188946-4189180 bp of the genome (GI No. 194447306) or a part of the complementary strand thereof. Wherein the salmonella genome-specific base sequence refers to a base sequence that is unique to the salmonella genome only and is not contained in the genome of other microorganisms.
Wherein, the primer group capable of amplifying the specific base sequence of the salmonella genome comprises but is not limited to a primer group A, or any one group of primer groups with homology of 73.7 percent and more with a single sequence in the sequence of the primer group or the sequence of the complementary strand thereof.
Primer set a:
upstream outer primer F3_ a: 5'-AGGCTTTAAAAAGGCCATG-3' (SEQ ID NO: 1);
downstream outer primer B3_ a: 5'-TAACGAACACTAACAGCAG-3' (SEQ ID NO: 2);
upstream inner primer FIP _ A: 5'-TTTTCGCTTCGCCTTGCTTAATGCCAAACAGGATAAAACC-3' (SEQ ID NO: 3);
the downstream inner primer BIP _ A: 5'-AGGAAGACGCTAAAAGCCAACGCTAAAACCGATATCAAAC-3' (SEQ ID NO: 4).
In the present invention, the primer set capable of amplifying the specific base sequence of the salmonella genome may further include a primer set having a homology of 73.7% 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, the following primer set B:
primer set B:
upstream outer primer F3_ B: 5'-AAAAACTCGGTTCCATCG-3' (SEQ ID NO: 5);
downstream outer primer B3_ B: 5'-AACACTAACAGCAGTTCG-3' (SEQ ID NO: 6) (73.7% homology to primer B3_ A: 5'-TAACGAACACTAACAGCAG-3');
upstream inner primer FIP _ B: 5'-GACTGGTTTTATCCTGTTTGGCGCGTCTATCAAAGGCTTT-3' (SEQ ID NO: 7);
the downstream inner primer BIP _ B: 5'-GCTAAATCTATCGCGGATAAGCAACGGATTATACCTGCTCTT-3' (SEQ ID NO: 8).
In the method of the present invention, the primer set capable of amplifying the specific base sequence of the salmonella genome may include, but is not limited to, a single loop primer. Preferably, the loop primer is one, and comprises loop primers LF or LB. The primer group capable of amplifying the specific base sequence of the salmonella genome is selected from any one of the following primer groups A 'and B'; or any one of the primer sets having a homology of 73.7% or more with a single sequence in the sequences of the primer sets A ', B' or the complementary strand sequences thereof:
primer set a':
upstream outer primer F3_ a: 5'-AGGCTTTAAAAAGGCCATG-3', respectively;
downstream outer primer B3_ a: 5'-TAACGAACACTAACAGCAG-3', respectively;
upstream inner primer FIP _ A: 5'-TTTTCGCTTCGCCTTGCTTAATGCCAAACAGGATAAAACC-3', respectively;
the downstream inner primer BIP _ A: 5'-AGGAAGACGCTAAAAGCCAACGCTAAAACCGATATCAAAC-3', respectively; upstream loop primer LF _ a: 5'-CGGTAAAATCAGCGTCCTGA-3' (SEQ ID NO: 9);
a primer set B':
upstream outer primer F3_ B: 5'-AAAAACTCGGTTCCATCG-3', respectively;
downstream outer primer B3_ B: 5'-AACACTAACAGCAGTTCG-3', respectively;
upstream inner primer FIP _ B: 5'-GACTGGTTTTATCCTGTTTGGCGCGTCTATCAAAGGCTTT-3', respectively;
the downstream inner primer BIP _ B: 5'-GCTAAATCTATCGCGGATAAGCAACGGATTATACCTGCTCTT-3', respectively;
downstream loop primer LB _ B: 5'-GGCGAAGCGAAAAAGGAAGA-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 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 image shows a characteristic step-shaped strip, the sample to be detected is positive to salmonella and contains salmonella; and if the electrophoretogram does not present a characteristic step-shaped strip, the sample to be detected is negative to salmonella. 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 salmonella and contains salmonella; if no turbidity is found, the sample to be detected is negative to salmonella. Or the bottom of the reaction tube can be observed by naked eyes after centrifugation to see whether the sediment exists or not, if the sediment exists at the bottom of the reaction tube, the sample to be detected is positive to salmonella and contains salmonella; if no precipitate is left at the bottom of the reaction tube, the sample to be detected is negative to salmonella.
The color development detection is to add color development reagent, including but not limited to calcein (50 μ M) or SYBRGreen I (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 salmonella; and if the color after the reaction is green, the sample to be detected is positive to salmonella and contains salmonella. When hydroxyl naphthol blue is used as a color developing agent, if the color after reaction is violet, the sample to be detected is salmonella negative; and if the color after the reaction is sky blue, the sample to be detected is positive for salmonella. The color development detection can be carried out in real time or at the end point by a detection instrument besides observing the reaction result by 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 salmonella negative; 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 salmonella. 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 systemAdding 0.6-1mM [ Mn ] into chlorophyll simultaneously2+]For example, 0.6-1mM MnCl2。
The invention also provides a primer used in the method for detecting the salmonella strain at constant temperature. The primer comprises a primer group capable of amplifying a specific base sequence of a salmonella genome, and the sequence of the primer includes but is not limited to a part of a nucleic acid sequence of 4188946-4189180 bp position of the salmonella genome with GI number of 194447306 or a part of a complementary strand thereof.
Wherein the primer group capable of amplifying the specific base sequence of the salmonella genome is selected from any one of the following primer groups, or is selected from any one of the primer groups with homology of 73.7% or more with a single sequence in the sequence of each primer group or the sequence of the complementary strand thereof. Wherein, the primer group includes but is not limited to the following primer group A. The primer set having a homology of 73.7% 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'-AGGCTTTAAAAAGGCCATG-3', respectively;
downstream outer primer B3_ a: 5'-TAACGAACACTAACAGCAG-3', respectively;
upstream inner primer FIP _ A: 5'-TTTTCGCTTCGCCTTGCTTAATGCCAAACAGGATAAAACC-3', respectively;
the downstream inner primer BIP _ A: 5'-AGGAAGACGCTAAAAGCCAACGCTAAAACCGATATCAAAC-3', respectively;
primer set B:
upstream outer primer F3_ B: 5'-AAAAACTCGGTTCCATCG-3', respectively;
downstream outer primer B3_ B: 5'-AACACTAACAGCAGTTCG-3', respectively;
upstream inner primer FIP _ B: 5'-GACTGGTTTTATCCTGTTTGGCGCGTCTATCAAAGGCTTT-3', respectively;
the downstream inner primer BIP _ B: 5'-GCTAAATCTATCGCGGATAAGCAACGGATTATACCTGCTCTT-3' are provided.
In the primer used in the method for detecting salmonella at constant temperature, the primer group capable of amplifying the specific base sequence of the salmonella genome can also comprise, but is not limited to, a loop primer; preferably, the loop primer is one, including LF or LB. The primer group capable of amplifying the specific base sequence of the salmonella 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 73.7% or more homology to 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'-AGGCTTTAAAAAGGCCATG-3', respectively;
downstream outer primer B3_ a: 5'-TAACGAACACTAACAGCAG-3', respectively;
upstream inner primer FIP _ A: 5'-TTTTCGCTTCGCCTTGCTTAATGCCAAACAGGATAAAACC-3', respectively;
the downstream inner primer BIP _ A: 5'-AGGAAGACGCTAAAAGCCAACGCTAAAACCGATATCAAAC-3', respectively;
upstream loop primer LF _ a: 5'-CGGTAAAATCAGCGTCCTGA-3', respectively;
a primer set B':
upstream outer primer F3_ B: 5'-AAAAACTCGGTTCCATCG-3', respectively;
downstream outer primer B3_ B: 5'-AACACTAACAGCAGTTCG-3', respectively;
upstream inner primer FIP _ B: 5'-GACTGGTTTTATCCTGTTTGGCGCGTCTATCAAAGGCTTT-3', respectively;
the downstream inner primer BIP _ B: 5'-GCTAAATCTATCGCGGATAAGCAACGGATTATACCTGCTCTT-3', respectively;
downstream loop primer LB _ B: 5'-GGCGAAGCGAAAAAGGAAGA-3' are provided.
The invention also provides a kit used in the method for detecting the salmonella strain at constant temperature, which comprises the primer group capable of amplifying the specific base sequence of the salmonella genome. In the kit, the primer group capable of amplifying the specific base sequence of the salmonella genome comprises but is not limited to a part of a nucleic acid sequence of 4188946-4189180 bp of the genome (GI No. 194447306) or a part of a complementary strand thereof as the primer sequence; the primer includes, but is not limited to, the primer set A and the like. But not limited to, a primer group having a homology of 73.7% or more with a single sequence in the aforementioned primer sequence or its complementary strand sequence; including but not limited to primer set B, etc.
In the kit of the present invention, the primer set capable of amplifying the specific base sequence of the salmonella genome may include, but is not limited to, a single loop primer; the loop primer serves as an optional component. Preferably, the loop primer is one, including LF or LB. The primer set comprising the loop primer LF 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 or LB loop primer. In one embodiment, the sequences of the primer sets are FIP, BIP, F3, B3, LF or FIP, BIP, F3, B3, LB primers, or primers with 73.7% or more homology to the aforementioned 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 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 BstDNA 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 also comprises a positive control template. In a specific embodiment, the positive control template includes, but is not limited to, salmonella whole genomic DNA, partial genomic DNA, or a vector comprising salmonella 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.
In the kit of the present invention,also included are color developers including, but 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 salmonella specificity, 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 primer of primer set A, vector pBR322-B containing any one primer of primer set B, vector pBR322-B 'containing any one primer of primer set B' … …, and the like. 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 salmonella.
The invention also provides application of the kit in constant-temperature detection of salmonella.
The invention also provides application of the vector in constant-temperature detection of salmonella.
The invention provides a simple, rapid and sensitive method for detecting salmonella, 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 salmonella 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 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 Salmonella detection method of example 7 of the present invention.
FIG. 2 shows the sensitivity of the Salmonella 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 Salmonella 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 salmonella strain for detection is from China Industrial microbial culture Collection management center, with the number of CICC 10420. 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/OD280The concentration was 1.8 and 408 ng/. mu.L.
(2) The salmonella genome DNA to be detected is taken as a template, self-prepared kits (shown in table 2 and table 3) are respectively adopted, a reaction system is prepared according to the conditions in table 3, and a salmonella specific amplification primer group is taken as a primer to carry out constant-temperature amplification reaction. The primers 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 the specific segment of the salmonella 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 salmonella.
Example 7 Salmonella-specific detection
26 strains (1 to 9 and 14 to 30 in Table 4 and FIG. 1) other than Salmonella were collected, cultured separately from the Salmonella strains (10 to 13 in Table 4 and FIG. 1), 1mL of the bacterial solution was taken, and bacterial DNA was extracted using kit IA, and 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 detection results are shown in table 4 and fig. 1, in fig. 1, 1 to 9 are staphylococcus aureus, staphylococcus aureus aureobasidium, staphylococcus epidermidis, rhodococcus equi, bacillus cereus, bacillus mycoides, listeria monocytogenes, listeria inokii and listeria ehelii, 14 to 30 are shigella dysenteriae, shigella baumannii, shigella flexneri, escherichia coli (containing clostridium botulinum type a gene), pathogenic escherichia coli, enterotoxigenic escherichia coli, escherichia hemorrhagic escherichia coli, clostridium sakazakii, yersinia enterocolitica, yersinia pseudotuberculosis, vibrio vulnificus, vibrio parahaemolyticus, vibrio freundii, vibrio cholerae O1 group and shigella sonnei, respectively: and negative controls 10-13 are salmonella enterica subspecies, salmonella enteritidis, salmonella typhimurium and salmonella paratyphi B respectively. In FIG. 1, only the products after the amplification reaction of Salmonella strains are bright green and positive results are shown in tubes 10-13. And the products of other non-salmonella strains and the negative control amplification reaction are orange, which are negative results, as shown in No. 1-9 and No. 14-30 tubes 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 present invention have good Salmonella strain specificity, i.e., only Salmonella strains are positive in amplification, and other non-Salmonella strains are negative.
Preparing a detection kit, wherein the primers adopted in the kit are respectively a primer group B, a primer group A 'and a primer group B', and the same detection results are respectively obtained according to the specific detection method, namely, the products after the amplification reaction of the non-salmonella strains and the negative control are negative results, and the products after the amplification reaction of the salmonella strains are positive results.
In addition, theoretical analysis was performed on the specificity of the primer sets a-B and a '-B' respectively according to the method described in table 1, and the results found that, under the condition that each primer allows at most 2 mismatches, at most two primers in each primer set were simultaneously aligned to non-salmonella bacteria, indicating that the specificity of each primer set was better.
Example 8 sensitivity detection
DNA of the bacterium CICC10420 was extracted by the method of example 2, and added to the reaction system using the kit IIB in a gradient of 50pg, 5pg, 500fg, 50fg and 5fg DNA, and LAMP amplification (primer set A) and visualization by adding a color-developing agent were carried out respectively under the other reaction conditions according to the method of example 2 of Table 3. As shown in fig. 2, 1 to 5 are 50pg, 5pg, 500fg, 50fg and 5fg, respectively, NTC: and (5) negative control. In FIG. 2, the reaction product of 50pg treatment appeared bright green and was a positive result, and the reaction products of 5pg, 500fg, 50fg and 5fg treatments and the negative control appeared orange and was a negative result. The results of the tests showed that the reaction tube contained a minimum of 50pg (about 10 pg equivalent) per reaction tube4Individual bacteria) can be detected.
According to the detection method, the DNA as low as 50 pg-500 fg in each reaction tube can be detected by using the primer group B and the primer groups A '-B' respectively according to the other steps and conditions.
Example 9 commonality testing
According to the method of example 7, salmonella enterica subspecies, salmonella enteritidis, salmonella typhimurium and salmonella paratyphi b (10-13 in table 4 and fig. 1) are respectively cultured and extracted with DNA, LAMP amplification (primer set is a) is performed, the detection results are shown in table 4 and fig. 1, the products after amplification reaction of the four salmonella strains are all bright green, and are positive results, which indicates that the primer set has good versatility.
Preparing a detection kit, wherein the primers adopted in the kit are respectively a primer group B, a primer group A 'and a primer group B', and the same detection results are respectively obtained according to the universal detection method, namely the four salmonella strains are amplified positively, which shows that the universality of each primer group is better.
According to the method described in table 1, theoretical analysis is performed on the universality of the primer groups a to B and the primer groups a 'to B', and the results show that the primer regions of the primer groups are completely matched with 38 salmonella strains, and can be theoretically used for the detection of the 38 salmonella strains, which indicates that the universality of the primer groups is better.
TABLE 1 analysis of the primer versatility and specificity in the existing detection methods for Salmonella
Note: a) carrying out Bowtie alignment on the sequence between primers F3 and B3 in the patent and the whole genome sequence of 38 GI strains of salmonella, and determining the position of a detection region in a genome; and performing Blast comparison on the detection region sequences in public database resources, wherein the primer regions are completely matched and have good universality. b) 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 cannot be simultaneously compared with the non-salmonella strains, the specificity is good.
TABLE 2 types and main components of kit for isothermal detection of salmonella
TABLE 3 examples 1 to 6 reaction conditions and results of the isothermal Salmonella detection method of the present invention
TABLE 4 strains used in the test and the results
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.
Claims (5)
1. A rapid isothermal salmonella detection method for non-diagnostic purposes, comprising the steps of:
(1) extracting genome DNA from a sample to be detected;
(2) 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 base sequence of the salmonella genome as a primer;
(3) determining whether salmonella exists in the sample to be detected or not by judging whether the reaction result is positive or not;
wherein the salmonella genome specific alkali sequence is a 4188946-4189180 bp bit sequence of the salmonella genome with GI number of 194447306;
wherein the primer group capable of amplifying the specific base sequence of the salmonella genome is a primer group A or a primer group A';
primer set a:
upstream outer primer F3_ a: 5'-AGGCTTTAAAAAGGCCATG-3' (SEQ ID NO: 1);
downstream outer primer B3_ a: 5'-TAACGAACACTAACAGCAG-3' (SEQ ID NO: 2);
upstream inner primer FIP _ A: 5'-TTTTCGCTTCGCCTTGCTTAATGCCAAACAGGATAAAACC-3' (SEQ ID NO: 3);
the downstream inner primer BIP _ A: 5'-AGGAAGACGCTAAAAGCCAACGCTAAAACCGATATCAAAC-3' (SEQ ID NO: 4);
primer set a':
upstream outer primer F3_ a: 5'-AGGCTTTAAAAAGGCCATG-3', respectively;
downstream outer primer B3_ a: 5'-TAACGAACACTAACAGCAG-3', respectively;
upstream inner primer FIP _ A: 5'-TTTTCGCTTCGCCTTGCTTAATGCCAAACAGGATAAAACC-3', respectively;
the downstream inner primer BIP _ A: 5'-AGGAAGACGCTAAAAGCCAACGCTAAAACCGATATCAAAC-3', respectively;
upstream loop primer LF _ a: 5'-CGGTAAAATCAGCGTCCTGA-3' (SEQ ID NO: 9).
2. The method of claim 1, wherein in step (2), the enzymatic reaction system comprises: 1 × BstDNA polymerase reaction bufferFlushing with 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, with or without 0.4-1.0. mu. mol/L LF _ A primer.
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 according to claim 1, wherein the primer is a primer set capable of amplifying a specific nucleotide sequence of the salmonella genome, the sequence of which is a part of a nucleic acid sequence of 4188946 to 4189180bp positions of the salmonella genome having GI number 194447306 or a part of a complementary strand thereof;
wherein the primer group capable of amplifying the specific base sequence of the salmonella genome is a primer group A or a primer group A';
primer set a:
upstream outer primer F3_ a: 5'-AGGCTTTAAAAAGGCCATG-3', respectively;
downstream outer primer B3_ a: 5'-TAACGAACACTAACAGCAG-3', respectively;
upstream inner primer FIP _ A: 5'-TTTTCGCTTCGCCTTGCTTAATGCCAAACAGGATAAAACC-3', respectively;
the downstream inner primer BIP _ A: 5'-AGGAAGACGCTAAAAGCCAACGCTAAAACCGATATCAAAC-3', respectively;
primer set a':
upstream outer primer F3_ a: 5'-AGGCTTTAAAAAGGCCATG-3', respectively;
downstream outer primer B3_ a: 5'-TAACGAACACTAACAGCAG-3', respectively;
upstream inner primer FIP _ A: 5'-TTTTCGCTTCGCCTTGCTTAATGCCAAACAGGATAAAACC-3', respectively;
the downstream inner primer BIP _ A: 5'-AGGAAGACGCTAAAAGCCAACGCTAAAACCGATATCAAAC-3', respectively;
upstream loop primer LF _ a: 5'-CGGTAAAATCAGCGTCCTGA-3' (SEQ ID NO: 9).
5. Use of a primer for isothermal salmonella detection for non-diagnostic purposes, wherein the primer is according to claim 4.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010017854.6A CN111073986B (en) | 2016-08-30 | 2016-08-30 | Rapid constant-temperature detection method for salmonella, primer group and application |
| CN201610767608.6A CN106367493B (en) | 2016-08-30 | 2016-08-30 | Method, primer and application for rapid constant-temperature detection of salmonella |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610767608.6A CN106367493B (en) | 2016-08-30 | 2016-08-30 | Method, primer and application for rapid constant-temperature detection of salmonella |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010017854.6A Division CN111073986B (en) | 2016-08-30 | 2016-08-30 | Rapid constant-temperature detection method for salmonella, primer group and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106367493A CN106367493A (en) | 2017-02-01 |
| CN106367493B true CN106367493B (en) | 2020-02-18 |
Family
ID=57901689
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610767608.6A Active CN106367493B (en) | 2016-08-30 | 2016-08-30 | Method, primer and application for rapid constant-temperature detection of salmonella |
| CN202010017854.6A Active CN111073986B (en) | 2016-08-30 | 2016-08-30 | Rapid constant-temperature detection method for salmonella, primer group and application |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010017854.6A Active CN111073986B (en) | 2016-08-30 | 2016-08-30 | Rapid constant-temperature detection method for salmonella, primer group and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN106367493B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106978503A (en) * | 2017-05-03 | 2017-07-25 | 宁夏出入境检验检疫局检验检疫综合技术中心 | A kind of LAMP detections primer sets and detection method for being used to detect salmonella |
| CN107022638A (en) * | 2017-06-05 | 2017-08-08 | 温和心 | The LAMP primer group of quick detection salmonella and its application |
| CN109402271A (en) * | 2017-08-14 | 2019-03-01 | 中国科学院微生物研究所 | For detecting the primer system and kit and their application and the method for detecting salmonella of salmonella |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102424842A (en) * | 2011-12-21 | 2012-04-25 | 中国人民解放军疾病预防控制所 | Salmonella LAMP (loop-mediated isothermal amplification) detection method, and special primer and kit thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101343660A (en) * | 2008-08-19 | 2009-01-14 | 华南理工大学 | In situ fluorescence ring mediated fast detecting reagent kit for salmonella and its use method |
| CN101880709B (en) * | 2009-12-29 | 2013-01-09 | 重庆市畜牧科学院 | Salmonella enteritidis detection reagent kit and method based on loop-mediated isothermal amplification technology |
| DE102012217646B3 (en) * | 2012-09-27 | 2013-10-24 | BCD Baltic Customized Diagnostics GmbH | Method for generating a set of samples for a detection method for Salmonella enterica ssp. enterica |
-
2016
- 2016-08-30 CN CN201610767608.6A patent/CN106367493B/en active Active
- 2016-08-30 CN CN202010017854.6A patent/CN111073986B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102424842A (en) * | 2011-12-21 | 2012-04-25 | 中国人民解放军疾病预防控制所 | Salmonella LAMP (loop-mediated isothermal amplification) detection method, and special primer and kit thereof |
Non-Patent Citations (2)
| Title |
|---|
| Efficient and Specific Detection of Salmonella in Food Samples Using a stn-Based Loop-Mediated Isothermal Amplification Method;Mevaree Srisawat等;《BioMed Research International》;20151231;第2.1-2.4、2.7和3.1节 * |
| NC_011083;Friche，W.F.等;《GenBank》;20160707;ORIGINAL * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106367493A (en) | 2017-02-01 |
| CN111073986A (en) | 2020-04-28 |
| CN111073986B (en) | 2022-09-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106434886B (en) | Method for rapidly detecting yersinia pseudotuberculosis at constant temperature, primer and application | |
| CN106367493B (en) | Method, primer and application for rapid constant-temperature detection of salmonella | |
| CN106367501B (en) | Method, primer and kit for rapid constant-temperature detection of salmonella | |
| CN106434887B (en) | Method, primer and kit for rapidly detecting staphylococcus aureus at constant temperature | |
| CN111004856B (en) | Rapid constant-temperature detection method, primer group and kit for vibrio vulnificus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211228 Address after: 200032 Shanghai Xuhui District Xietu Road No. 2140 Patentee after: Shanghai Institute of biomedical technology Address before: 201203 Shanghai city Pudong New Area Keyuan Road No. 1278 Patentee before: SHANGHAI CENTER FOR BIOINFORMATION TECHNOLOGY |
|
| TR01 | Transfer of patent right |