CN111073989B - Rapid constant-temperature detection method and application of shigella nucleic acid - Google Patents

Abstract

The invention discloses a rapid constant temperature detection method for shigella, a primer set and application thereof. The method comprises the following steps: extracting genome DNA from a sample to be detected; taking the genome DNA as a template, taking a primer group capable of amplifying a shigella specific sequence as a primer, and performing isothermal amplification reaction under an enzyme reaction system; and determining whether shigella exists in the sample to be detected by judging whether the reaction result is positive. The detection method has the advantages of high sensitivity, high specificity, short detection time, simple result judgment, convenient operation, low cost and wide application prospect.

Description

Rapid constant-temperature detection method and application of shigella nucleic acid

The application is filed 8/30/2016, and has the application number of 201610767703.6 and the invention name of: the divisional application of Chinese invention patent application of a method, a primer and a kit for detecting shigella at a rapid constant temperature; the parent application claims priority of Chinese patent application with application date of 2015, 9 and 2, application number of 201510556917.4 and the invention name of 'method, primer and kit for rapidly detecting Cronobacter sakazakii at constant temperature'.

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 shigella at constant temperature.

Background

Shigella spp is a gram-negative enterobacteria, the most common pathogenic bacteria for human bacillary dysentery, known as Shigella dysenteriae. Shigella bacteria can be transmitted through contaminated water and food. The shigella bacteria are highly susceptible to the shigella bacteria, and the shigella bacteria can cause acute and middle-toxic bacillary dysentery in infants, have serious illness and high death rate. Therefore, the shigella in water or food can be detected timely and accurately, which not only provides basis for effectively preventing, treating and controlling water or food borne infectious diseases, but also is a necessary means for guaranteeing the health of people.

The traditional shigella bacteria detection method has the defects of relatively long detection period, relatively complex operation and relatively low detection efficiency, and is difficult to meet the requirements of the modern society on high flux, high sensitivity, high specificity, rapidness and convenience in the food-borne pathogenic bacteria detection process. In recent years, with the development of nucleic acid molecule detection technology, researchers have developed detection means such as PCR and real-time fluorescence PCR, but both methods require special detection instruments, and thus are not suitable for being widely applied to basic detection departments, especially real-time in-situ detection performed inside an enterprise production line. In order to ensure food safety, a rapid, simple, accurate method for detecting shigella 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 (comprising upstream and downstream outer primers F3 and B3 and upstream and downstream inner primers FIP and BIP, wherein FIP consists of F1C and F2, and BIP consists of B1C and B2) for 6 regions of a target sequence, and uses a DNA polymerase with strand displacement activity to perform a nucleic acid amplification reaction by incubating for about 60min under isothermal conditions, thereby generating macroscopic reaction byproducts-white magnesium pyrophosphate precipitate (see Notomi T, okayama H, masubechi H, yonekawa T, watanabe K, amino N, hase T.Loop-mediated isothermal amplification of DNA, nucleic Acids Research,2000jun 15;28 (12): E63). The technology has the advantages of no need of a PCR instrument or a fluorescent quantitative PCR instrument, completion at constant temperature, capability of judging the reaction result by naked eyes, high sensitivity, strong specificity, short reaction time, convenient operation, low cost and the like.

Primer design is the most critical step in the LAMP technology, and conventionally, a recognized specific gene of a certain organism to be detected is introduced into an online website (http:// primerexplorer.jp/e) of the LAMP primer design, and related parameters are set to generate a primer set. That is, the user must first ensure that the target gene is a specific sequence of the species to be tested. Taking patent ZL201010145716.2 and ZL201310737377.0 as examples, the specific genes of Shigella reported in the literature, i.e. ipaH genes, are respectively used for detecting the Shigella by adopting the LAMP technology. However, so-called "putative specific genes" are often based on a hysteresis knowledge and are not necessarily updated based on growing microbial genome data, resulting in that primers obtained based on the target gene sequences do not necessarily ensure their versatility and/or specificity in practical applications. The present invention shows the problem that the primer versatility in the prior art cannot be ensured by using Table 1. That is, the shigella detection sequences used in the prior art methods are not actually common to shigella, i.e., there is a potential for missing a portion of the strain of shigella. Similar problems exist in the validation of the versatility, i.e., there is a possibility that non-shigella may be mistakenly identified as shigella. Therefore, a shigella detection method capable of ensuring specificity and universality is needed in the industry, meanwhile, the requirements of basic detection departments on rapidness and convenience are met, and real-time in-situ detection can be conveniently carried out 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 prior LAMP technology, fully utilizes the abundant genome sequence information of microorganisms in the current public data resources and corresponding sequence analysis tools, designs a primer group for specifically identifying shigella, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention provides a method for detecting shigella by rapid isothermal amplification, a primer group and a kit based on the design of shigella LAMP primer by microbial genome data resources (data of 5 days of 8 months of 2013) in a GenBank database. The detection method for detecting shigella has the advantages of high sensitivity, high specificity, short detection time, simple result judgment, convenient operation and low cost.

The invention provides a method for rapidly detecting shigella strains, which comprises 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 a shigella genome specific base sequence as a primer, and performing isothermal amplification reaction under an enzyme reaction system;

(3) And determining whether shigella exists in the sample to be detected by judging whether the reaction result is positive.

The method for detecting shigella strains at constant temperature comprises the steps of extracting genome DNA from a sample to be detected, taking the genome DNA as a template, taking a shigella specific amplification primer group as a primer, performing a constant-temperature amplification reaction, and then determining whether shigella exists in the sample to be detected by judging whether a reaction result is positive. Wherein the enzyme reaction system includes, but is not limited to, a DNA polymerase reaction system.

In the invention, the specific base sequence of the shigella genome is 1921404 ～ 1921811bp of the shigella with the GI number of 110804074.

In the present invention, the primer set capable of amplifying a shigella genome-specific base sequence is a part of a 1921404 ～ 1921811bp nucleic acid sequence of the genome (GI number 110804074) or a part of the complementary strand thereof. Wherein, the shigella genome specific base sequence refers to a base sequence which is unique to only shigella genome and not contained in other microbial genomes.

Wherein, the primer group capable of amplifying the specific base sequence of the shigella genome comprises, but is not limited to, a primer group A, or any one of the primer groups with 75% or more of the single sequence homology with the primer group sequence or the complementary strand sequence thereof.

Primer group a:

upstream outer primer f3_a:5'-AGTCAGTCTGGATATGGGCC-3' (SEQ ID NO: 1);

downstream outer primer b3_a:5'-AAGAGTGATTTCCTGGCCTG-3' (SEQ ID NO: 2);

upstream inner primer FIP_A:5'-ACGCCCTGAGACAACAGAACCGGGTGTGATAAATGGGGCC-3' (SEQ ID NO: 3);

downstream inner primer bip_a:5'-GGATGCTGAGCACCCCTTCAAACGTGAATATTCCGTTGCTGGC-3' (SEQ ID NO: 4).

In the present invention, the primer set capable of amplifying a specific base sequence of a shigella genome may further include a primer set having 75% or more homology with a single sequence of the aforementioned primer set sequences or the complementary strand sequences thereof, and the primer set includes, but is not limited to, the following primer set B:

primer group B:

upstream outer primer f3_b:5'-AGTCAGTCTGGATATGGGCC-3' (SEQ ID NO: 5);

downstream outer primer b3_b:5'-TGATTTCCTGGCCTGGGTAA-3' (SEQ ID NO: 6) (75% homology to primer B3_A 5'-AAGAGTGATTTCCTGGCCTG-3')

Upstream inner primer FIP_B:5'-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3' (SEQ ID NO: 7);

downstream inner primer bip_b:5'-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3' (SEQ ID NO: 8).

In the method of the present invention, the primer set capable of amplifying a shigella genome-specific base sequence may include, but is not limited to, a loop primer. Preferably, the loop primer is one, namely loop primer LB. The primer group capable of amplifying the shigella genome specific base sequence is selected from any one of the following primer groups A ', B'; or any one selected from the group consisting of primer sets having 75% or more homology to a single sequence of the primer sets A ', B' or the complementary strand sequences thereof:

primer set a':

upstream outer primer f3_a:5'-AGTCAGTCTGGATATGGGCC-3';

downstream outer primer b3_a:5'-AAGAGTGATTTCCTGGCCTG-3';

upstream inner primer FIP_A:5'-ACGCCCTGAGACAACAGAACCGGGTGTGATAAATGGGGCC-3'; downstream inner primer bip_a:5'-GGATGCTGAGCACCCCTTCAAACGTGAATATTCCGTTGCTGGC-3';

downstream loop primer lb_a:5'-GTGTGTCGGGTATGATGATGCCG-3' (SEQ ID NO: 9);

primer set B':

upstream outer primer f3_b:5'-AGTCAGTCTGGATATGGGCC-3';

downstream outer primer b3_b:5'-TGATTTCCTGGCCTGGGTAA-3';

upstream inner primer FIP_B:5'-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3'; downstream inner primer bip_b:5'-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3';

downstream loop primer lb_b:5'-GTGTGTCGGGTATGATGATGCCG-3' (SEQ ID NO: 10).

In one embodiment (loop-containing primer) of the method of the invention, the isothermal amplification enzymatic reaction system 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 primer, 0.15-0.3 mu mol/L F3 and B3 primer, 0.4-1.0 mu mol/L LB primer, 0.16-0.64U/. Mu.L Bst DNA polymerase and 0-1.5mol/L betaine. In another specific embodiment (without loop primer), the isothermal amplification enzyme reaction system 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 primer, 0.15-0.3 mu mol/L F3 and B3 primer, 0.16-0.64U/. Mu.L Bst DNA polymerase and 0-1.5mol/L betaine. The loop primer contributes to an improvement in reaction efficiency. For example, 1 XBst DNA polymerase reaction buffer may be 1 XThermopol reaction buffer containing 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl,10mmol/L (NH 4) ₂ SO4，0.1％Triton X-100，2mM MgSO ₄ . MgSO in 1 XBst DNA polymerase reaction buffer ₄ And magnesium ion Mg in an enzyme reaction system ²⁺ And (5) performing merging treatment.

In the method of the invention, the reaction program of the isothermal amplification reaction is (1) incubation for 10-90 min, preferably 10-60 min at 60-65 ℃; (2) the reaction is stopped for 2 to 20 minutes at the temperature of 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 development detection, and the like. 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 chart shows a characteristic ladder-shaped strip, the sample to be detected shows shigella positivity and contains shigella; if the electrophoresis chart does not show a characteristic ladder-shaped strip, the sample to be tested is shigella negative. The turbidity detection is to detect turbidity by naked eyes or a turbidity meter, and if turbidity occurs in a detection tube, a sample to be detected is shigella positive and contains shigella; if no turbidity is found, the sample to be tested is shigella negative. Or observing whether the reaction tube bottom has sediment or not by naked eyes after centrifugation, and if the reaction tube bottom has sediment, the sample to be tested is shigella positive and contains shigella; if the bottom of the reaction tube has no sediment, the sample to be tested is shigella negative.

The color development test is to add a color developing agent including but not limited to calcein (50. Mu.M) or SYBR Green I (30-50X), or hydroxynaphthol blue (i.e., HNB, 120-150. Mu.M) into the reaction tube. When calcein or SYBR Green I is adopted as a color developing agent, if the color after the reaction is orange, the sample to be detected is shigella negative; if the color after the reaction is green, the sample to be detected is positive to shigella and contains shigella. When hydroxyl naphthol blue is adopted as a color-developing agent, if the color after the reaction is violet, the sample to be detected is shigella negative; if the color after the reaction is sky blue, the sample to be tested is shigella positive. Besides the reaction result observed by naked eyes, the color development detection can also be carried out by detecting the reaction result in real time or through an end point by a detection instrument, and 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 shigella negative by reasonably setting the threshold value of the negative reaction; and when the reaction result of the sample to be detected is greater than the threshold value, the sample to be detected is shigella positive. Such detection instruments include, but are not limited to, fluorescence spectrophotometers, fluorescent quantitative PCR instruments, isothermal amplification microfluidic chip nucleic acid analyzers, genie II isothermal amplification fluorescent detection systems, and the like.

In the color development detection, if calcein or hydroxynaphthol blue is adopted as the color developing agent, the calcein or hydroxynaphthol blue can be added before the isothermal amplification reaction, or after the isothermal amplification reaction is finished, preferably before the isothermal amplification reaction, so that the possibility of reaction pollution can be effectively reduced. If SYBR Green I is used as the developer, it is added after the isothermal amplification reaction is complete. If calcein is used as the color-developing agent, 50. Mu.M calcein and 0.6-1mM Mn are added to the enzyme reaction system ²⁺ ]For example, 0.6-1mM MnCl ₂ 。

The invention also provides a primer used in the method for detecting the shigella strain at constant temperature. The primer includes a primer set capable of amplifying a specific base sequence of the shigella genome, including, but not limited to, a portion of the 1921404 ～ 1921811bp nucleic acid sequence of the shigella genome with GI number 110804074 or a portion of the complementary strand thereof.

Wherein the primer set capable of amplifying the shigella genome-specific base sequence is selected from any one of the following primer sets, or from any one of the primer sets having 75% or more homology with a single sequence in the sequence of each primer set or the complementary strand sequence thereof. Wherein the primer set includes, but is not limited to, the following primer set A. The primer set having 75% or more homology to 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 group a:

upstream outer primer f3_a:5'-AGTCAGTCTGGATATGGGCC-3';

downstream outer primer b3_a:5'-AAGAGTGATTTCCTGGCCTG-3';

upstream inner primer FIP_A:5'-ACGCCCTGAGACAACAGAACCGGGTGTGATAAATGGGGCC-3'; downstream inner primer bip_a:5'-GGATGCTGAGCACCCCTTCAAACGTGAATATTCCGTTGCTGGC-3';

primer group B:

upstream outer primer f3_b:5'-AGTCAGTCTGGATATGGGCC-3';

downstream outer primer b3_b:5'-TGATTTCCTGGCCTGGGTAA-3';

upstream inner primer FIP_B:5'-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3';

downstream inner primer bip_b:5'-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3'.

The primer group capable of amplifying the shigella genome specific base sequence can also comprise, but is not limited to, a loop primer; preferably, the loop primer is one and LB. The primer group capable of amplifying the shigella genome specific base sequence is selected from any one of the following primer groups A ', B'; or any one selected from the group consisting of primer sets having 75% or more homology to a single sequence of the primer sets A ', B' or the complementary strand sequences thereof:

primer set a':

upstream outer primer f3_a:5'-AGTCAGTCTGGATATGGGCC-3';

downstream outer primer b3_a:5'-AAGAGTGATTTCCTGGCCTG-3';

upstream inner primer FIP_A:5'-ACGCCCTGAGACAACAGAACCGGGTGTGATAAATGGGGCC-3'; downstream inner primer bip_a:5'-GGATGCTGAGCACCCCTTCAAACGTGAATATTCCGTTGCTGGC-3';

downstream loop primer lb_a:5'-GTGTGTCGGGTATGATGATGCCG-3';

primer set B':

upstream outer primer f3_b:5'-AGTCAGTCTGGATATGGGCC-3';

downstream outer primer b3_b:5'-TGATTTCCTGGCCTGGGTAA-3';

upstream inner primer FIP_B:5'-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3';

downstream inner primer bip_b:5'-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3';

downstream loop primer lb_b:5'-GTGTGTCGGGTATGATGATGCCG-3'.

The invention also provides a kit for the method for detecting the shigella strain at constant temperature, which comprises the primer group capable of amplifying the specific base sequence of the shigella genome. In the kit of the present invention, the primer set capable of amplifying a shigella genome-specific base sequence includes, but is not limited to, a portion of a 1921404 ～ 1921811bp nucleic acid sequence of a genome (GI number: 110804074) or a portion of a complementary strand thereof as the primer sequence; the primer includes, but is not limited to, the primer set a. Also included are, but not limited to, primer sets having 75% or more homology to a single sequence of the aforementioned primer sequences or the complementary strand sequences thereof; including but not limited to primer set B.

In the kit of the present invention, the primer set capable of amplifying the shigella genome-specific base sequence may include, but is not limited to, a loop primer; the loop primer is used as an optional component. Preferably, the loop primer is one and LB. Primer sets comprising loop primer LB include, but are not limited to, primer sets A ', B', and the like. In a specific embodiment, the kit of the present invention may contain 0.4 to 1.0. Mu. Mol/L of LB loop primer. In one embodiment, the primer set has a sequence of FIP, BIP, F3, B3, LB, or a primer having 75% or more homology with a single primer of the foregoing sequence or the complementary strand sequence thereof.

The kit also comprises Bst DNA polymerase buffer solution, bst DNA polymerase, dNTP solution and Mg ²⁺ (MgSO ₄ Or MgCl ₂ ) And one or more of betaines. In one embodiment, the enzyme reaction system of the kit of the invention comprises 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 primer, 0.15-0.3 mu mol/L F3 and B3 primer, 0.16-0.64U/. Mu.L Bst DNA polymerase and 0-1.5mol/L betaine. For example, 1 XBst DNA polymerase reaction buffer may be 1 XThermopol reaction buffer containing 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl,10mmol/L (NH 4) ₂ SO4，0.1％Triton X-100，2mM MgSO ₄ . MgSO in 1 XBst DNA polymerase reaction buffer ₄ And enzyme reactionMagnesium ion Mg in the system ²⁺ And (5) performing merging treatment.

The kit also comprises a positive control template. In a specific embodiment, the positive control template includes, but is not limited to, whole genomic DNA, partial genomic DNA of shigella, or a vector comprising whole genomic DNA or partial genomic DNA of shigella.

The kit of the invention further comprises a negative control template, wherein 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 to, 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 vector comprising any one of the primer sets A, B, A ', B'. The vector contains a DNA sequence with shigella specificity, so that the vector can be applied to the research fields of microbiology, 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 chromosomal 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, 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'. A vector lambda phage-A comprising any one of the primers of the primer set A, a vector lambda phage-B comprising any one of the primers of the primer set B, a vector lambda phage-A comprising any one of the primers of the primer set A ', a vector lambda phage-B ' comprising any one of the primers of the primer set B ', etc.

The invention also provides application of the primer selected from any one of the primer groups A, B, A ', B' in isothermal detection of shigella.

The invention also provides application of the kit in constant temperature detection of shigella.

The invention also provides application of the carrier in constant temperature detection of shigella.

The invention provides a simple, rapid and sensitive method for detecting shigella, 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 shigella 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 existing common detection method, the constant-temperature amplification method can be carried out under the constant-temperature condition, only a simple constant-temperature device is needed, expensive instruments in a PCR experiment are not needed, and steps such as electrophoresis detection are not needed on amplified products, so that the method is very suitable for being widely applied to popularization and use in various communities including basic food safety detection departments, and can be fully applied even under the environment with relatively insufficient molecular biology expertise and skill foundation. The above preferred conditions may be arbitrarily combined based on the common knowledge in the art, and all the conditions fall within the scope of the present invention.

Drawings

FIG. 1 shows the specificity of the constant temperature detection method for Shigella according to example 7 of the present invention.

FIG. 2 shows the sensitivity of the shigella 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, to which the present invention is not limited. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims. The procedures, conditions, reagents, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for those specifically mentioned below, and the present invention is not particularly limited.

EXAMPLES 1-6 Shigella 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 shigella (shigella flexneri) strain used for detection is derived from China general microbiological culture collection center, and the number is CGMCC 1.1868. Extracting genome DNA and DNA OD from 1mL bacterial culture with bacterial nucleic acid extraction kit of Beijing Tiangen bioengineering company ₂₆₀ /OD ₂₈₀ 1.8 at a concentration of 400 ng/. Mu.L.

(2) The method comprises the steps of taking shigella genome DNA to be detected as a template, respectively adopting self-matched kits (see table 2 and table 3), preparing a reaction system according to the conditions shown in table 3, and carrying out isothermal amplification reaction by taking shigella specific amplification primer groups as primers. The primers in examples 1 to 6 are primer sets A, A, A ', B, B', respectively.

(3) The amplification results were confirmed by electrophoresis detection, turbidity detection or chromogenic detection under the conditions described 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 shigella and obtain the detection result. In addition, when the detection is performed by using the detector, the reaction time is shortened to 10min, and the detection effect is also good (example 6). Therefore, the invention can be applied to detecting whether shigella is contained in a sample.

EXAMPLE 7 Shigella specific detection

Non-shigella strain 26 (1 to 13, 17 to 29 in table 4 and fig. 1) was collected, these strains and shigella strains (14 to 16 in table 4 and fig. 1) were cultured, 1mL of bacterial liquid was taken, bacterial DNA was extracted using kit IA, and LAMP amplification (primer set a) and observation with addition of a color developer were performed, respectively, with reference to the reaction system and conditions of example 1.

As shown in Table 4 and FIG. 1, in FIG. 1, 1 to 13 are Staphylococcus aureus, staphylococcus aureus subspecies aureobacteria, staphylococcus epidermidis, rhodococcus equi, bacillus cereus, bacillus mycoides, listeria monocytogenes, listeria incarnata, listeria, salmonella enterica subspecies enterica, salmonella typhimurium, salmonella paratyphi B, and 17 to 29 are Escherichia coli (containing clostridium botulinum type A gene), pathogenic Escherichia coli, diarrhea causing Escherichia coli, enterotoxigenic Escherichia coli, hemorrhagic Escherichia coli, cronobacter sakazakii, enterocolitis, yersinia pseudotuberculosis, vibrio vulnificus, vibrio parahaemolyticus, vibrio and Vibrio cholerae, NTC: and negative control, wherein 14-16 are shigella dysenteriae, shigella sonnei and shigella flexneri respectively. In FIG. 1, only the products after the shigella strain amplification reaction appear bright green, as positive results, as shown in lanes 14-16. The other non-shigella strains and the products after the negative control amplification reaction are all orange, and are negative results, such as the tubes 1 to 13, 17 to 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 present invention have good shigella strain specificity, i.e., only shigella strains amplify positive, and other non-shigella 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 respectively obtained according to the specific detection method, namely, the products after amplification reaction of the non-shigella strain and the negative control are negative results, and the products after amplification reaction of the shigella strain are positive results.

In addition, according to the method described in Table 1, the specificity of each of the primer set A, the primer set B and the primer sets A ', B' was theoretically analyzed, and as a result, it was found that, in the case that each primer allowed at most three mismatches, at most one primer was compared to non-shigella, indicating that the specificity of each primer set was good.

Example 8 sensitivity detection

The DNA of Shigella flexneri CGMCC 1.1868 was extracted as in example 2, using kit IIB, and DNA gradients of 10ng, 1ng, 100pg, 10pg, 1pg, 100fg and 10fg were added to the reaction system, and other reaction conditions were observed by LAMP amplification (primer set A) and addition of a color-developer, respectively, as in example 2 of Table 3. As shown in fig. 2, 1 to 7 are 10ng, 1ng, 100pg, 10pg, 1pg, 100fg and 10fg, respectively, ntc: negative control. In FIG. 2, the reaction products of the 10ng and 1ng treatments appear bright green as positive results, and the reaction products of the 100pg, 10pg, 1pg, 100fg, 10fg treatments and the negative control appear orange as negative results. The results of the test showed that each reaction tube contained a minimum of 1ng (approximately equivalent to 2X 10 ⁵ Bacteria) can still be detected.

According to the above detection method, other steps and conditions are the same, and the DNA as low as 1 ng-1 pg in each reaction tube can still be detected by using the primer group B and the primer groups A ', B'.

Example 9 commonality test

According to the method of example 1, shigella dysenteriae, shigella sonnei and shigella flexneri (14 to 16 in table 4 and fig. 1) were cultured and DNA was extracted, respectively, LAMP amplification was performed (primer set a) and the detection results are shown in table 4 and fig. 1, and the products after the three shigella dysenteriae amplification reactions were all shown as bright green, and were positive results, indicating that the primer set was better in versatility.

Preparing a detection kit, wherein the primers adopted in the kit are respectively a primer group B and a primer group A ', and the primer groups B' respectively obtain the same detection results according to the above general detection method, namely three shigella strains are positive in amplification, which indicates that the general purpose of each primer group is good.

According to the method shown in Table 1, the primer region of each primer group is completely matched with 8 shigella strains (GI numbers are 30061571, 74310614, 82542618, 110804074, 187730020, 344915202, 377520096 and 384541581 respectively) by theoretical analysis on the universality of the primer group A, the primer group B and the primer group A ', and the primer group B', and the primer region of each primer group is 1-2 mismatched with the shigella dysenteriae with the GI number 82775382 in the primer region (B3 and/or B2), so that the primer group can be theoretically used for detecting the 9 shigella strains, and the universality of each primer group is good.

TABLE 1 general and specific analysis of primers in existing detection methods of Shigella

Note that: a) The sequences between primers F3 and B3 of the patent were aligned with 9 genomes of Shigella (GI numbers 110804074, 82775382, 30061571, 74310614, 82542618, 187730020, 344915202, 377520096 and 384541581, respectively) to determine the location of the detection region in the GI number 110804074 genome. b) And Blast comparison is carried out on the detection region sequences in public database resources, and primer regions are completely matched to be good in universality. c) Blast comparison is carried out on the detection region sequences in public database resources, and 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-shigella strains, the specificity is good.

TABLE 2 kit types and main composition for isothermal detection of Shigella

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TABLE 3 examples 1-6 reaction conditions and results of the method for isothermal detection of Shigella according to the present invention

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Table 4 strains used in the experiments and the results of the tests

Note that: a) CGMCC: china general microbiological culture Collection center, CICC: china center for type culture Collection (CMCC): china medical bacterial culture Collection center. b) ++. Positive result, -: negative results.

<110> Shanghai technology institute, shanghai Wangwang food group Co., ltd

<120> shigella nucleic acid rapid constant temperature detection method and application

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<210> 1

<211> 20

<212> DNA

<213> artificial sequence

<400> 1

agtcagtctg gatatgggcc 20

<210> 2

<211> 20

<212> DNA

<213> artificial sequence

<400> 2

aagagtgatt tcctggcctg 20

<210> 3

<211> 40

<212> DNA

<213> artificial sequence

<400> 3

acgccctgag acaacagaac cgggtgtgat aaatggggcc 40

<210> 4

<211> 43

<212> DNA

<213> artificial sequence

<400> 4

ggatgctgag caccccttca aacgtgaata ttccgttgct ggc 43

<210> 5

<211> 20

<212> DNA

<213> artificial sequence

<400> 5

agtcagtctg gatatgggcc 20

<210> 6

<211> 20

<212> DNA

<213> artificial sequence

<400> 6

tgatttcctg gcctgggtaa 20

<210> 7

<211> 40

<212> DNA

<213> artificial sequence

<400> 7

acgccctgag acaacagaac ccgggtgtga taaatggggc 40

<210> 8

<211> 43

<212> DNA

<213> artificial sequence

<400> 8

ggatgctgag caccccttca aacctgccag gtgaatattc cgt 43

<210> 9

<211> 23

<212> DNA

<213> artificial sequence

<400> 9

gtgtgtcggg tatgatgatg ccg 23

<210> 10

<211> 23

<212> DNA

<213> artificial sequence

<400> 10

gtgtgtcggg tatgatgatg ccg 23

Claims (9)

1. A rapid isothermal detection method for shigella for non-diagnostic purposes, comprising the steps of:

(1) Extracting genome DNA from a sample to be detected;

(2) Taking the genome DNA as a template, taking a primer group capable of amplifying a shigella genome specific base sequence as a primer, and performing isothermal amplification reaction under an enzyme reaction system;

(3) Determining whether shigella exists in the sample to be detected by judging whether the reaction result is positive;

wherein, the specific base sequence of the shigella genome is 1921404 ～ 1921811bp of the shigella genome with the GI number of 110804074;

wherein, the primer group capable of amplifying the shigella genome specific base sequence is a primer group B or a primer group B';

primer group B:

upstream outer primer f3_b:5'-AGTCAGTCTGGATATGGGCC-3' (SEQ ID NO: 5);

downstream outer primer b3_b:5'-TGATTTCCTGGCCTGGGTAA-3' (SEQ ID NO: 6);

upstream inner primer FIP_B:5'-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3' (SEQ ID NO: 7);

downstream inner primer bip_b:5'-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3' (SEQ ID NO: 8);

primer set B':

upstream outer primer f3_b:5'-AGTCAGTCTGGATATGGGCC-3';

downstream outer primer b3_b:5'-TGATTTCCTGGCCTGGGTAA-3';

upstream inner primer FIP_B:5'-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3'; downstream inner primer bip_b:

5’-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3’；

downstream loop primer lb_b:5'-GTGTGTCGGGTATGATGATGCCG-3' (SEQ ID NO: 10).

2. The method of claim 1, wherein in step (2), the enzyme reaction system comprises: 1 XBstDNA polymerase reaction buffer, 2-9mmol/LMg ²⁺ 1.0-1.6mmol/LdNTP, 0.8-2.0. Mu. Mol/L FIP_B and BIP_B primers, 0.15-0.3. Mu. Mol/L F3_B and B3_B primers, 0.16-0.64U/. Mu.LBstDNA polymerase, 0-1.5mol/L betaine, including or not including 0.4-1.0. Mu. Mol/L LB_B primer.

3. The method of claim 1, wherein the isothermal amplification reaction is performed by a reaction sequence comprising: (1) incubating for 10-90 min at 60-65 ℃; (2) the reaction is stopped for 2 to 20 minutes at the temperature of 80 ℃.

4. A primer for rapid isothermal detection of shigella, characterized in that the primer is a primer group capable of amplifying a shigella genome specific base sequence, and the sequence is a part of 1921404 ～ 1921811bp nucleic acid sequence of shigella genome with GI number 110804074 or a part of complementary strand thereof;

wherein, the primer group capable of amplifying the shigella genome specific base sequence is a primer group B or a primer group B';

primer group B:

upstream outer primer f3_b:5'-AGTCAGTCTGGATATGGGCC-3' (SEQ ID NO: 5);

downstream outer primer b3_b:5'-TGATTTCCTGGCCTGGGTAA-3' (SEQ ID NO: 6);

upstream inner primer FIP_B:5'-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3' (SEQ ID NO: 7);

downstream inner primer bip_b:5'-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3' (SEQ ID NO: 8);

primer set B':

upstream outer primer f3_b:5'-AGTCAGTCTGGATATGGGCC-3';

downstream outer primer b3_b:5'-TGATTTCCTGGCCTGGGTAA-3';

upstream inner primer FIP_B:5'-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3'; downstream inner primer bip_b:

5’-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3’；

downstream loop primer lb_b:5'-GTGTGTCGGGTATGATGATGCCG-3' (SEQ ID NO: 10).

5. A rapid isothermal detection kit for shigella comprising the primer of claim 4.

6. The kit of claim 5, further comprising BstDNA polymerase reaction buffer, bstDNA polymerase, dNTP solution, mg ²⁺ One or more of betaine.

7. The kit of claim 5, wherein the enzyme reaction system of the kit comprises: 1 XBstDNA polymerase reaction buffer, 2-9mmol/LMg ²⁺ 1.0-1.6mmol/LdNTP, 0.8-2.0. Mu. Mol/L FIP_B and BIP_B primers, 0.15-0.3. Mu. Mol/L F3_B and B3_B primers, including or not including 0.4-1.0. Mu. Mol/L LB_B primer, 0.16-0.64U/. Mu.LBSTDNA polymerase, and 0-1.5mol/L betaine.

8. Use of a primer for the isothermal detection of shigella for non-diagnostic purposes, characterized in that the primer is according to claim 4.

9. Use of a kit according to any one of claims 5 to 7 for the isothermal detection of shigella for non-diagnostic purposes.

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