CN111088377B

CN111088377B - Rapid constant temperature detection method for staphylococcus aureus, primer set and application

Info

Publication number: CN111088377B
Application number: CN202010018178.4A
Authority: CN
Inventors: 李园园; 刘伟; 李雪玲; 贾犇; 韦朝春; 陆长德; 李亦学
Original assignee: Shanghai Institute Of Biomedical Technology
Current assignee: Shanghai Institute Of Biomedical Technology
Priority date: 2016-08-30
Filing date: 2016-08-30
Publication date: 2023-05-23
Anticipated expiration: 2036-08-30
Also published as: CN106434887B; CN111088377A; CN106434887A

Abstract

The invention discloses a rapid constant temperature detection method for staphylococcus aureus, a primer group 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 staphylococcus aureus specific sequence as a primer, and performing isothermal amplification reaction under an enzyme reaction system; and determining whether staphylococcus aureus 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 for staphylococcus aureus, primer set and application

The application is filed 8/30/2016, and has the application number of 201610767557.7 and the invention name of: a method, primer and kit for quickly detecting staphylococcus aureus at constant temperature are applied separately from the Chinese patent application.

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 staphylococcus aureus at constant temperature.

Background

Staphylococcus aureus (Staphylococcus aureus) belongs to the genus staphylococcus, and is a gram-positive bacterium in the form of a sphere. Staphylococcus aureus is the most common pathogenic bacteria in human suppurative infection, and can cause local suppurative infection, pneumonia, pseudomembranous enteritis, pericarditis and the like, and even systemic infection such as septicemia, sepsis and the like. Meanwhile, enterotoxins produced by the method can pollute food to cause food poisoning, and bring serious public health burden to human beings. Therefore, it is very important for the prevention and detection of the bacterium.

At present, the traditional culture method is generally adopted for detecting staphylococcus aureus internationally, but the detection period is longer, the operation is relatively complex, the detection efficiency is lower, and the requirements of the modern society on high flux, high sensitivity, high specificity, rapidness and convenience in the food-borne pathogenic bacteria detection process are difficult to meet. With the development of nucleic acid molecule detection technology in recent years, researchers have developed detection means of PCR and fluorescence PCR technologies, but these two methods require special detection instruments, and are therefore not suitable for being widely applied to real-time in-situ detection performed in basic detection departments, especially in the interior of enterprise production lines. To ensure food safety, a rapid, simple, and accurate method for detecting staphylococcus aureus 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 the invention patent CN 101701252B and CN 101880711A as examples, the invention adopts the LAMP technology to detect staphylococcus aureus aiming at nuc gene and clfA gene which are specific genes of staphylococcus aureus reported in literature. 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 versatility of the prior art cannot be ensured by table 1. That is, the staphylococcus aureus detection sequences used in the prior art methods are not actually common to staphylococcus aureus, i.e., there is a potential for missing a portion of the strain of staphylococcus aureus. Similar problems exist with regard to specificity validation, i.e., there is a potential for the non-staphylococcus aureus to be mistakenly identified as staphylococcus aureus. Therefore, there is a need in the industry for a staphylococcus aureus detection method that can ensure specificity and universality, and simultaneously meet the requirements of the basic detection department on rapidness and convenience, and can conveniently perform real-time in-situ detection in the 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 sequence information of the microorganism genome and the corresponding sequence analysis tools which are rich in the current public data resources, designs a primer group for specifically identifying staphylococcus aureus, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention provides a method for detecting staphylococcus aureus by rapid isothermal amplification, a primer group and a kit based on the design of a staphylococcus aureus LAMP primer by using a microorganism genome data resource (data of 8 months and 5 days in 2013) in a GenBank database. The detection method for detecting staphylococcus aureus 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 staphylococcus aureus 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 specific base sequence of staphylococcus aureus genome as a primer, and performing isothermal amplification reaction under an enzyme reaction system;

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

The invention discloses a method for detecting staphylococcus aureus strains at constant temperature, which comprises the steps of extracting genome DNA from a sample to be detected, taking the genome DNA as a template, taking a staphylococcus aureus specific amplification primer group as a primer, carrying out a constant-temperature amplification reaction, and then determining whether staphylococcus aureus 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 staphylococcus aureus genome is 2645869 ～ 2646117bp of staphylococcus aureus with the GI number of 148266447.

In the present invention, the primer set capable of amplifying a genome-specific base sequence of staphylococcus aureus is a part of a 2645869 ～ 2646117bp nucleic acid sequence of the genome (GI number 148266447) or a part of a complementary strand thereof. Wherein, the staphylococcus aureus genome specific base sequence refers to a base sequence which is only unique to the staphylococcus aureus genome and not contained in other microorganism genomes.

Wherein the primer set capable of amplifying a specific base sequence of a staphylococcus aureus genome comprises, but is not limited to, a primer set A, or any one of the primer sets having a homology of 68% or more with a single sequence in the primer set sequence or the complementary strand sequence thereof.

Primer group a:

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

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

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

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

In the present invention, the primer set capable of amplifying a specific base sequence of a staphylococcus aureus genome may further comprise a primer set having a homology of 68% or more with a single sequence of the aforementioned primer set sequences or the complementary strand sequences thereof, the primer set including but not limited to the following primer set B:

primer group B:

upstream outer primer f3_b:5'-ATGACTAAAGCCACATCCA-3' (SEQ ID NO: 5) (homology 68% with primer F3_A 5'-CTAAAGCCACATCCAATATAGG-3');

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

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

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

In the method of the present invention, the primer set capable of amplifying a genome-specific base sequence of staphylococcus aureus may or may not contain a loop primer. The loop primer may be one or more, including primers LF and/or LB. The primer group capable of amplifying the staphylococcus aureus genome specific base sequence is a primer group A'; or any one selected from the group consisting of a primer group having 68% or more homology to a single sequence in the primer group A' sequence or the complementary strand sequence thereof:

primer set a':

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

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

upstream inner primer FIP_A:5'-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3';

downstream inner primer bip_a:5'-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3';

upstream loop primer lf_a:5'-TCCCTTTAATTAAGTAAACCC-3' (SEQ ID NO: 9);

and/or, a downstream loop primer lb_a:5'-TAAGAACTAGTTAGTGACTA-3' (SEQ ID NO: 10).

In particular embodiments, for example, the primer set a' may comprise only one upstream loop primer, only one downstream loop primer, or both an upstream loop primer and a downstream loop primer.

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 LF and/or LB primer, 0.16-0.64U/. Mu.L Bst DNA polymerase and 0-1.5mol/L betaine. In another embodiment (without loop primer), the isothermal amplification is performed enzymaticallyThe reaction system is as follows: 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 is positive to staphylococcus aureus and contains staphylococcus aureus; if the electrophoresis chart does not show a characteristic ladder-shaped strip, the sample to be tested is negative to staphylococcus aureus. The turbidity detection is to detect turbidity by naked eyes or a turbidity meter, and the sample to be detected is positive to staphylococcus aureus and contains staphylococcus aureus when obvious turbidity appears in a detection tube; if no turbidity is found, the sample to be tested is negative to staphylococcus aureus. 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 positive to staphylococcus aureus and contains staphylococcus aureus; if the bottom of the reaction tube has no sediment, the sample to be tested is negative to staphylococcus aureus.

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 staphylococcus aureus negative; if the color after the reaction is green, the sample to be detected is positive to staphylococcus aureus and contains staphylococcus aureus. 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 staphylococcus aureus negative; if the color after the reaction is sky blue, the sample to be tested is positive to staphylococcus aureus. 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 a detecting 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 staphylococcus aureus negative by reasonably setting the threshold value of the negative reaction; and when the reaction result of the sample to be tested is greater than the threshold value, the sample to be tested is positive to staphylococcus aureus. 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 primers for use in a method of isothermal detection of staphylococcus aureus strains. The primer includes a primer set capable of amplifying a specific base sequence of a staphylococcus aureus genome, including, but not limited to, a portion of a 2645869 ～ 2646117bp nucleic acid sequence of a staphylococcus aureus genome with a GI number of 148266447 or a portion of a complementary strand thereof.

Wherein the primer set capable of amplifying a genome-specific base sequence of Staphylococcus aureus is selected from any one of the following primer sets, or from any one of the primer sets having a homology of 68% or more 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, primer set a. The primer set having a homology of 68% or more 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'-CTAAAGCCACATCCAATATAGG-3';

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

upstream inner primer FIP_A:5'-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3';

primer group B:

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

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

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

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

The primer group capable of amplifying the specific base sequence of the staphylococcus aureus genome can also comprise or not comprise one or more loop primers; the loop primer is LF and/or LB. The primer group capable of amplifying the staphylococcus aureus genome specific base sequence is a primer group A'; or any one selected from the group consisting of a primer group having 68% or more homology to a single sequence in the primer group A' sequence or the complementary strand sequence thereof:

primer set a':

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

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

upstream inner primer FIP_A:5'-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3';

upstream loop primer lf_a:5'-TCCCTTTAATTAAGTAAACCC-3';

and/or, a downstream loop primer lb_a:5'-TAAGAACTAGTTAGTGACTA-3'.

In specific embodiments, the primer set a' 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 those shown as FIP, BIP, F3, B3, LF and LB, respectively, or primers having 68% or more homology with a single primer in the aforementioned primer sequence or the complementary strand sequence thereof.

The invention also provides a kit for the method for detecting staphylococcus aureus strains at constant temperature, which comprises the primer group capable of amplifying specific base sequences of staphylococcus aureus genome. In the kit of the present invention, the primer set capable of amplifying a genome-specific base sequence of Staphylococcus aureus includes, but is not limited to, a part of a 2645869 ～ 2646117bp nucleic acid sequence of a genome (GI number: 148266447) or a part 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 68% 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 invention, the primer set capable of amplifying a genome-specific base sequence of staphylococcus aureus may or may not comprise one or more loop primers; the loop primer is used as an optional component. The loop primer is LF and/or LB. Primer sets comprising loop primers LF and/or LB include, but are not limited to, primer set a'. 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 primer set has the sequences of FIP, BIP, F3, B3, LF and LB, respectively, or a primer having 68% or more homology with a single primer of the foregoing sequences or the complementary strand sequences 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 magnesium ion Mg in an enzyme reaction 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, or a vector comprising whole genomic DNA or partial genomic DNA of staphylococcus aureus.

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 primers selected from the primer set A, B, A'. The vector contains a DNA sequence with staphylococcus aureus 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 microorganism 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'. 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', etc.

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

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

The invention also provides application of the carrier in detecting staphylococcus aureus at constant temperature.

The invention provides a simple, rapid and sensitive method for detecting staphylococcus aureus, 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 staphylococcus aureus 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 isothermal detection method for Staphylococcus aureus according to example 7 of the present invention.

FIG. 2 shows the sensitivity of the method for detecting Staphylococcus aureus 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 Staphylococcus aureus 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 staphylococcus aureus strain used for detection is derived from China industry microbiological culture collection center, and is numbered CICC21600 (ATCC 27217). 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 50.4 ng/. Mu.L.

(2) The genome DNA of staphylococcus aureus to be detected is used as a template, self-matched kits (see table 2 and table 3) are respectively adopted, a reaction system is prepared according to the conditions shown in table 3, and a staphylococcus aureus specific amplification primer group is used as a primer for carrying out isothermal amplification reaction. The primers in examples 1 to 6 were primer sets A, A '(2 loop primers), A' (1 loop primer), B, and 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 staphylococcus aureus 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 staphylococcus aureus is contained in a sample.

EXAMPLE 7 Staphylococcus aureus specific assay

Non-staphylococcus aureus 27 strains (3-29 in table 4 and fig. 1) were collected, these strains and staphylococcus aureus strains (1 and 2 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, 3 to 29 are respectively Staphylococcus epidermidis, rhodococcus equi, bacillus cereus, bacillus mycoides, listeria monocytogenes, listeria inobacter, listeria, salmonella enterica subspecies enterica, salmonella typhimurium, salmonella paratyphi, shigella dysenterica, shigella Boshi, shigella flexneri, escherichia coli (containing clostridium botulinum type A gene), pathogenic Escherichia coli, diarrhea-causing Escherichia coli, enterotoxigenic Escherichia coli, hemorrhagic Escherichia coli, cronobacter sakazakii, yersinia enterocolitica, yersinia pseudotuberculosis, vibrio vulnificus, vibrio parahaemolyticus, vibrio febrio and cholera, NTC: negative control, 1-2 are staphylococcus aureus, staphylococcus aureus subspecies golden yellow. In FIG. 1, only the products after the amplification reaction of Staphylococcus aureus strains appear bright green, as positive results, as shown in lanes 1-2. The other non-staphylococcus aureus strains and the products after the negative control amplification reaction are all orange, and are negative results, such as the tubes 3-29 and the NTC negative control tube.

As can be seen from the results of FIG. 1 and Table 4, the detection kit and the detection method of the present invention have good staphylococcus aureus strain specificity, namely, only staphylococcus aureus strains amplify positively, and other non-staphylococcus aureus strains are negative.

Preparing a detection kit, wherein the primers adopted in the kit are respectively a primer group B and a primer group A', and respectively obtaining the same detection results according to the specific detection method, namely, the products after amplification reaction of non-staphylococcus aureus strains and negative control are negative results, and the products after amplification reaction of staphylococcus aureus strains are positive results.

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

Example 8 sensitivity detection

The DNA of the bacterium CICC21600 was extracted as in example 1, and the LAMP amplification (primer set A) and the addition of the color-developing reagent were performed under the other reaction conditions according to the procedure of example 1 of Table 3, respectively, using the kit IB, and adding the DNA in the reaction system in a gradient of 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10 fg. As shown in FIG. 2, 1-7 are 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, NTC, respectively: negative control. In FIG. 2, the reaction products treated with 10ng, 1ng, 100pg appear bright green as positive results, and the reaction products treated with 10pg, 1pg, 100fg, 10fg and negative controls appear orange as negative results. The results of the test showed that each reaction tube contained a minimum of 100pg (approximately equivalent to 3X 10 ⁴ Bacteria) can be detected.

According to the above detection method, other steps and conditions are the same, and the DNA as low as 100pg to 1pg in each reaction tube can still be detected by using the primer group B and the primer group A'.

Example 9 commonality test

According to the method shown in Table 1, the primer groups A-B and the primer group A' are subjected to theoretical analysis, and the result shows that the primer region of each primer group is completely matched with 43 staphylococcus aureus strains in a database, and can be theoretically used for detecting the 43 staphylococcus aureus strains, so that the primer groups are good in universality.

TABLE 1 general and specific analysis of primers in the existing detection methods of Staphylococcus aureus

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Note that: a) The sequences between primers F3 and B3 of the patent were Bowtie aligned with 43 whole genomes of Staphylococcus aureus to determine the position of the detection region in the GI No. 148266447 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. The results of the general analysis on three genomes are only listed in this table. 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; can not be simultaneously compared with non-staphylococcus aureus, which indicates that the specificity is good.

TABLE 2 kit type and main composition for isothermal detection of Staphylococcus aureus

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

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

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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 bioinformatics research center

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<213> artificial sequence

<400> 5

atgactaaag ccacatcca 19

<210> 6

<211> 19

<212> DNA

<213> artificial sequence

<400> 6

atgccttaca ttgatgctg 19

<210> 7

<211> 40

<212> DNA

<213> artificial sequence

<400> 7

tcgttggtga gcaattgagg agtatcgttc cagatttgtg 40

<210> 8

<211> 43

<212> DNA

<213> artificial sequence

<400> 8

gactaggggt agtaatcatt ggccactttg ctatggaacg taa 43

<210> 9

<211> 21

<212> DNA

<213> artificial sequence

<400> 9

tccctttaat taagtaaacc c 21

<210> 10

<211> 20

<212> DNA

<213> artificial sequence

<400> 10

taagaactag ttagtgacta 20

Claims

1. A rapid isothermal detection method for staphylococcus aureus for non-diagnostic purposes, which is characterized by comprising the following steps:

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

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

(3) Determining whether staphylococcus aureus exists in the sample to be tested by judging whether the reaction result is positive;

wherein the specific base sequence of the staphylococcus aureus genome is 2645869 ～ 2646117bp bit sequence of a staphylococcus aureus genome with a GI number of 148266447;

wherein, the primer group capable of amplifying the specific base sequence of the staphylococcus aureus genome is a primer group B;

primer group B:

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

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

2. The method of claim 1, wherein the steps of(2) In the above, the enzyme reaction system comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg ²⁺ 1.0-1.6mmol/L dNTP,0.8-2.0 mu mol/L FIP_B and BIP_B primer, 0.15-0.3 mu mol/L F3_B and B3_B primer, 0.16-0.64U/mu L Bst DNA polymerase, 0-1.5mol/L betaine.

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 staphylococcus aureus, characterized in that the primer comprises a primer set capable of amplifying a staphylococcus aureus genome specific base sequence which is a part of a 2645869 ～ 2646117bp nucleic acid sequence of a staphylococcus aureus genome with a GI number of 148266447 or a part of a complementary strand thereof;

primer group B:

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

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

5. A rapid isothermal detection kit for staphylococcus aureus, characterized in that the kit comprises a primer group capable of amplifying a staphylococcus aureus genome specific base sequence;

primer group B:

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

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

6. The kit of claim 5, further comprising Bst DNA polymerase reaction buffer, bst DNA 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 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg ²⁺ 1.0-1.6mmol/L dNTP,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.L Bst DNA polymerase, and 0-1.5mol/L betaine.

8. Use of a primer for the isothermal detection of staphylococcus aureus 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 staphylococcus aureus for non-diagnostic purposes.