CN106434887B - Method, primer and kit for rapidly detecting staphylococcus aureus at constant temperature - Google Patents

Abstract

The invention discloses a method, a primer group and a kit for rapidly detecting staphylococcus aureus at constant temperature. The method comprises the following steps: extracting genome DNA from a sample to be detected; carrying out constant-temperature amplification reaction in an enzyme reaction system by taking the genome DNA as a template and a primer group capable of amplifying a specific sequence of staphylococcus aureus as a primer; and determining whether the sample to be detected has staphylococcus aureus or not by judging whether the reaction result is positive or not. The detection method has the advantages of high sensitivity and high specificity, short detection time, simple result judgment, convenient operation, low cost and wide application prospect.

Description

Method, primer and kit for rapidly detecting staphylococcus aureus at constant temperature

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method, primers 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 with spherical thallus. Staphylococcus aureus is the most common pathogenic bacterium in human pyogenic infection, and can cause local pyogenic infection, pneumonia, pseudomembranous enteritis, pericarditis and the like, and even septicemia, sepsis and other systemic infections. Meanwhile, the enterotoxin produced by the method can pollute food to cause food poisoning, and very serious public health burden is brought to human beings. Therefore, it is very important to prevent and detect the bacteria.

At present, the traditional culture method is generally adopted internationally for detecting staphylococcus aureus, but the detection period is longer, the operation is relatively complex, the detection efficiency is lower, and the requirements of high flux, high sensitivity, high specificity, rapidness and convenience in the detection process of food-borne pathogenic bacteria in modern society are difficult to meet. In recent years, with the development of nucleic acid molecule detection technology, researchers have developed detection means of PCR and fluorescence PCR technologies, but these two methods require special detection instruments, and are not suitable for real-time field detection in basic detection departments, especially in production lines of enterprises. In order to ensure the safety of food, 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 (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 patents CN 101701252B and CN 101880711A are taken as examples, and the detection of the staphylococcus aureus is carried out by adopting LAMP technology aiming at the nuc gene and clfA gene which are specific genes of the staphylococcus aureus reported in the literature. 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 staphylococcus aureus test sequence used in the prior art methods is not actually common to staphylococcus aureus, i.e., there is a possibility of missing some strains of staphylococcus aureus. A similar problem exists in the confirmation of specificity, i.e., the possibility of incorrectly identifying non-s.aureus as s.aureus. Therefore, a staphylococcus aureus detection method capable of ensuring specificity and universality is urgently needed in the industry, and meanwhile, the requirements of primary 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 primer design of the prior LAMP technology, fully utilizes abundant microbial genome sequence information in the current public data resources and corresponding sequence analysis tools, designs a primer group for specifically identifying staphylococcus aureus, 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 detecting staphylococcus aureus by rapid isothermal amplification, and relates to the design of staphylococcus aureus LAMP primers based on microbial genome data resources (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 and specificity, short detection time, simple result judgment, convenience in 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) 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 a specific base sequence of a staphylococcus aureus genome as a primer;

(3) and determining whether the sample to be detected has staphylococcus aureus or not by judging whether the reaction result is positive or not.

The method for detecting the staphylococcus aureus strain at constant temperature extracts genome DNA from a sample to be detected, takes the genome DNA as a template and a staphylococcus aureus specific amplification primer group as a primer to carry out constant temperature amplification reaction, and then determines whether the staphylococcus aureus exists in the sample to be detected by judging whether the reaction result is positive or not. Wherein, the enzyme reaction system includes but is not limited to DNA polymerase reaction system.

In the invention, the genome specific alkali sequence of the staphylococcus aureus is a bit sequence of 2645869-2646117 bp of the staphylococcus aureus with the GI number of 148266447.

In the present invention, the primer set capable of amplifying a genome-specific nucleotide sequence of Staphylococcus aureus is a part of a nucleic acid sequence of 2645869 to 2646117bp positions of the genome (GI No. 148266447) or a part of a complementary strand thereof. Wherein the staphylococcus aureus genome-specific base sequence is a base sequence which is unique to the staphylococcus aureus genome and is not contained in the genomes of other microorganisms.

Wherein the primer group capable of amplifying the specific base sequence of the staphylococcus aureus genome comprises but is not limited to a primer group A, or any one group selected from the primer group with 68 percent of homology or 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'-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);

the downstream inner primer BIP _ A: 5'-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3' (SEQ ID NO: 4).

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

primer set B:

upstream outer primer F3_ B: 5'-ATGACTAAAGCCACATCCA-3' (SEQ ID NO: 5) (68% homology to primer F3_ A5'-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);

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

primer set a':

upstream outer primer F3_ a: 5'-CTAAAGCCACATCCAATATAGG-3', respectively;

downstream outer primer B3_ a: 5'-TATGCCTTACATTGATGCTG-3', respectively;

upstream inner primer FIP _ A: 5'-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3', respectively;

the downstream inner primer BIP _ A: 5'-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3', respectively;

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

and/or, the downstream loop primer LB _ A: 5'-TAAGAACTAGTTAGTGACTA-3' (SEQ ID NO: 10).

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

In a specific embodiment (including a loop primer), the enzyme reaction system for isothermal amplification is as follows: 1 XBst DNA polymerase reaction buffer, 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 and/or LB primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. In another embodiment (without loop primer), the enzyme reaction system for isothermal amplification is: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L 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 (pH 8.8), 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.

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 chart shows a characteristic stepped strip, the sample to be detected is staphylococcus aureus positive and contains staphylococcus aureus; and if the electrophoretogram does not present a characteristic stepped strip, the sample to be detected is staphylococcus aureus negative. The turbidity detection is to detect turbidity by naked eye observation or a turbidity meter, and if the detection tube is obviously turbid, the sample to be detected is staphylococcus aureus positive and contains staphylococcus aureus; if no turbidity is found, the sample to be detected is staphylococcus aureus negative. Or visually observing whether the bottom of the reaction tube has a precipitate after centrifugation, wherein if the bottom of the reaction tube has the precipitate, the sample to be detected is staphylococcus aureus positive and contains staphylococcus aureus; if no precipitate is left at the bottom of the reaction tube, the sample to be detected is staphylococcus aureus negative.

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 staphylococcus aureus negative; if the color after the reaction is green, the sample to be detected is staphylococcus aureus positive and contains staphylococcus aureus. When hydroxyl naphthol blue is used as a color developing agent, if the color after 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 detected is staphylococcus aureus positive. 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 staphylococcus aureus 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 to staphylococcus aureus. 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.

The above-mentionedIn the color development detection, if the calcein or hydroxynaphthol blue is used as the color development agent, the color development agent can be added before the isothermal amplification reaction, or can be added after the isothermal amplification reaction is completed, preferably before the isothermal 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 system, and 0.6-1mM [ Mn ] is added²⁺]For example, 0.6-1mM MnCl₂。

The invention also provides a primer used in the method for detecting the staphylococcus aureus strain at constant temperature. The primer comprises a primer group capable of amplifying a specific base sequence of a staphylococcus aureus genome, and the sequence of the primer is, but not limited to, a part of a nucleic acid sequence of 2645869-2646117 bp of the staphylococcus aureus genome with the GI number of 148266447 or a part of a complementary strand of the nucleic acid sequence.

Wherein the primer group capable of amplifying the genome-specific nucleotide sequence of Staphylococcus aureus is selected from any one of the following primer groups, or from any one of the primer groups having 68% or more homology with a single sequence in the sequences of the primer groups or the complementary strand sequences thereof. Wherein, the primer group includes but is not limited to primer group A. The primer set having 68% or more homology to a single sequence in the aforementioned primer set sequence or the sequence of the complementary strand thereof includes, but is not limited to, the following primer set B.

Primer set a:

upstream outer primer F3_ a: 5'-CTAAAGCCACATCCAATATAGG-3', respectively;

downstream outer primer B3_ a: 5'-TATGCCTTACATTGATGCTG-3', respectively;

upstream inner primer FIP _ A: 5'-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3', respectively;

the downstream inner primer BIP _ A: 5'-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3', respectively;

primer set B:

upstream outer primer F3_ B: 5'-ATGACTAAAGCCACATCCA-3', respectively;

downstream outer primer B3_ B: 5'-ATGCCTTACATTGATGCTG-3', respectively;

upstream inner primer FIP _ B: 5'-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3', respectively;

the downstream inner primer BIP _ B: 5'-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3' are provided.

In the primer used in the method for detecting staphylococcus aureus at constant temperature, 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 specific base sequence of the staphylococcus aureus genome is a primer group A'; or any one selected from the group consisting of primer sets having 68% or more homology to a single sequence in the sequence of the primer set A' or the sequence of the complementary strand thereof:

primer set a':

upstream outer primer F3_ a: 5'-CTAAAGCCACATCCAATATAGG-3', respectively;

downstream outer primer B3_ a: 5'-TATGCCTTACATTGATGCTG-3', respectively;

upstream inner primer FIP _ A: 5'-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3', respectively;

the downstream inner primer BIP _ A: 5'-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3', respectively;

upstream loop primer LF _ a: 5'-TCCCTTTAATTAAGTAAACCC-3', respectively;

and/or, the downstream loop primer LB _ A: 5'-TAAGAACTAGTTAGTGACTA-3' are provided.

In a specific embodiment, 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 a specific embodiment, the primers are respectively FIP, BIP, F3, B3, LF and LB primers or primers with 68% or more homology with single primer in the aforementioned primer sequence or its complementary strand sequence.

The invention also provides a kit used in the method for detecting the staphylococcus aureus strain at constant temperature, which comprises the primer group capable of amplifying the specific base sequence of the staphylococcus aureus genome. In the kit, the primer group capable of amplifying the genome-specific nucleotide sequence of the staphylococcus aureus comprises but is not limited to a part of a nucleic acid sequence at 2645869-2646117 bp of a genome (GI No. 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. But not limited to, a primer set having 68% or more homology to a single sequence in the aforementioned primer sequence or its complementary strand sequence; including but not limited to primer set B.

In the kit of the present invention, the primer set capable of amplifying a specific base sequence of a staphylococcus aureus genome may or may not comprise one or more loop primers; the loop primer serves as an optional component. The loop primer is LF and/or LB. 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 sequences of the primer sets are respectively the primers shown in FIP, BIP, F3, B3, LF and LB, or the primers with 68% or more homology to the single primer of 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 (pH 8.8), 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, whole genomic DNA, partial genomic DNA of staphylococcus aureus, 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, and the negative control template comprises but is not limited to double distilled water.

The kit 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 [ Mn²⁺]For example, MnCl₂。

The kit of the invention also comprises double distilled water.

The kit of the invention also comprises a nucleic acid extraction reagent.

The invention also provides a vector, which comprises any one primer selected from the primer group A, B, A'. The vector contains a DNA sequence with specificity of staphylococcus aureus, so that 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, and vector pBR322-A 'containing any one primer of primer set A'. A vector lambda phage-A containing any one primer of the primer set A, a vector lambda phage-B containing any one primer of the primer set B, a vector lambda phage-A 'containing any one primer of the primer set A', and the like.

The invention also provides application of any one primer selected from the primer group 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 constant temperature detection of staphylococcus aureus.

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, convenience in 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 Staphylococcus aureus detection method of example 7 of the present invention.

FIG. 2 shows the sensitivity of the Staphylococcus aureus 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 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 for detection is from China industrial microorganism culture collection management center, with the number CICC21600(ATCC 27217). 1mL of the bacterial culture was used to extract genomic DNA and DNA OD using a bacterial nucleic acid extraction kit from Beijing Tiangen bioengineering Co₂₆₀/OD₂₈₀At a concentration of 1.8, 50.4 ng/. mu.L.

(2) The method comprises the steps of taking the genomic DNA of staphylococcus aureus to be detected as a template, respectively adopting self-prepared kits (shown in tables 2 and 3), preparing a reaction system according to the conditions in the table 3, and taking a specific amplification primer group of the staphylococcus aureus as a primer to carry out constant-temperature amplification reaction. The primers in examples 1 to 6 were primer set A, A '(2-loop primer), A' (1-loop primer), B and 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 reaction system adopted by the detection method can well amplify the specific fragment of the staphylococcus aureus 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 staphylococcus aureus.

Example 7 Staphylococcus aureus-specific detection

27 strains of non-Staphylococcus aureus (3 to 29 in Table 4 and FIG. 1) were collected, cultured separately from the Staphylococcus aureus strains (1 and 2 in Table 4 and FIG. 1), 1mL of the bacterial suspension 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, 3 to 29 are staphylococcus epidermidis, rhodococcus equi, bacillus cereus, bacillus mycoides, listeria monocytogenes, listeria inokii, listeria ehelii, salmonella enterica, salmonella enteritidis, salmonella typhimurium, salmonella paratyphi b, shigella dysenteriae, shigella boydii, shigella flexneri, escherichia coli (containing clostridium botulinum type a gene), pathogenic escherichia coli, enterotoxin-producing escherichia coli, enterotoxigenic escherichia coli, enterohemorrhagic escherichia coli, escherichia sakazakii, yersinia enterocolitica, yersinia pseudotuberculosis, vibrio vulnificus, vibrio parahaemolyticus, vibrio and NTC cholera, respectively: and (5) negative controls, namely 1-2 staphylococcus aureus and staphylococcus aureus subspecies aurantiaca respectively. In FIG. 1, only the products of the amplification reaction of Staphylococcus aureus strains appeared bright green, and were positive results, as shown in tubes 1-2. And the products of other non-staphylococcus aureus strains and the negative control amplification reaction are orange, which is a negative result, as shown in tubes No. 3-29 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 specificity for Staphylococcus aureus strains, i.e., only Staphylococcus aureus strains are positive in amplification, 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 the same detection results are respectively obtained according to the specific detection method, namely, the products of the non-staphylococcus aureus strains and the negative control amplification reaction are negative results, and the products of the staphylococcus aureus strains after the amplification reaction are positive results.

In addition, theoretical analysis is performed on the specificity of the primer groups A-B and A' according to the method described in Table 1, and the result shows that under the condition that at most three mismatches are allowed for each primer, at most one primer in each primer group is compared with non-Staphylococcus aureus, which indicates that the specificity of each primer group is better.

Example 8 sensitivity detection

The DNA of bacterium CICC21600 was extracted according to the method of example 1,using the kit IB, the reaction system was subjected to LAMP amplification (primer set A) and visualization by adding a color-developing agent, respectively, according to the method of example 1 in Table 3, with the use of DNA gradients of 10ng, 1ng, 100pg, 10pg, 1pg, 100fg and 10 fg. As shown in fig. 2, 1-7 are 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, NTC: and (5) negative control. In FIG. 2, the reaction products of 10ng, 1ng and 100pg treatments appeared bright green and as positive results, the reaction products of 10pg, 1pg, 100fg and 10fg treatments and the negative control appeared orange and as negative results. The results of the tests showed that the reaction tube contained a minimum of 100pg (about 3X 10. sup. th equivalent) per reaction tube⁴Individual bacteria) can be detected.

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

Example 9 commonality testing

According to the method described in table 1, theoretical analysis is performed on the universality of the primer groups a to B and a primer group a', and the result shows that the primer region of each primer group is completely matched with 43 strains of staphylococcus aureus in the database, and can be theoretically used for detection of the 43 strains of staphylococcus aureus, which indicates that the universality of each primer group is better.

TABLE 1 analysis of primer versatility and specificity in existing detection methods for Staphylococcus aureus

Note: a) the sequence between primers F3 and B3 in the patent was 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 performing Blast comparison on the detection region sequences in public database resources, wherein the primer regions are completely matched and have good universality. The results of the commonality analysis are presented only on three genomes. c) Performing Blast comparison on the detection region sequence in public database resources, wherein the higher the matching degree of the primer region is, the worse the specificity is; the specificity is good as the result that the antibody can not be simultaneously compared with non-staphylococcus aureus.

TABLE 2 types and main components of kit for isothermal detection of staphylococcus aureus

TABLE 3 examples 1-6 reaction conditions and test results in the method for isothermal detection of Staphylococcus aureus according to 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 detection method for staphylococcus aureus for non-diagnostic purposes is characterized by comprising the following steps:

(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 taking a primer group capable of amplifying a specific base sequence of a staphylococcus aureus genome as a primer;

(3) determining whether the sample to be detected has staphylococcus aureus or not by judging whether the reaction result is positive or not;

wherein the staphylococcus aureus genome specific alkali sequence is a 2645869-2646117 bp bit sequence of a staphylococcus aureus genome with a GI (glycemic index) of 148266447;

wherein the primer group capable of amplifying the specific base sequence of the staphylococcus aureus genome is a primer group A or a primer group A';

primer set 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);

the downstream inner primer BIP _ A: 5'-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3' (SEQ ID NO: 4);

primer set a':

upstream outer primer F3_ a: 5'-CTAAAGCCACATCCAATATAGG-3', respectively;

downstream outer primer B3_ a: 5'-TATGCCTTACATTGATGCTG-3', respectively;

upstream inner primer FIP _ A: 5'-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3', respectively;

the downstream inner primer BIP _ A: 5'-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3', respectively;

upstream loop primer LF _ a: 5'-TCCCTTTAATTAAGTAAACCC-3' (SEQ ID NO: 9) and the downstream loop primer LB _ A: 5'-TAAGAACTAGTTAGTGACTA-3' (SEQ ID NO: 10).

2. The method of claim 1, wherein in step (2), the enzymatic reaction system comprises: 1 XBstDNA polymerase reaction buffer, 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, and 0.4-1.0. mu. mol/L LF _ A and/or LB _ A primers.

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 group capable of amplifying a specific base sequence of a staphylococcus aureus genome, and the sequence is a nucleic acid sequence with 2645869-2646117 bp sites of the staphylococcus aureus genome with GI number 148266447;

wherein the primer group capable of amplifying the specific base sequence of the staphylococcus aureus genome is a primer group A or a primer group A';

primer set a:

upstream outer primer F3_ a: 5'-CTAAAGCCACATCCAATATAGG-3', respectively;

downstream outer primer B3_ a: 5'-TATGCCTTACATTGATGCTG-3', respectively;

upstream inner primer FIP _ A: 5'-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3', respectively;

the downstream inner primer BIP _ A: 5'-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3', respectively;

primer set a':

upstream outer primer F3_ a: 5'-CTAAAGCCACATCCAATATAGG-3', respectively;

downstream outer primer B3_ a: 5'-TATGCCTTACATTGATGCTG-3', respectively;

upstream inner primer FIP _ A: 5'-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3', respectively;

the downstream inner primer BIP _ A: 5'-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3', respectively;

upstream loop primer LF _ a: 5'-TCCCTTTAATTAAGTAAACCC-3' and downstream loop primer LB _ A: 5'-TAAGAACTAGTTAGTGACTA-3', or both.

5. Use of a primer for isothermal detection of staphylococcus aureus, not for diagnostic purposes, according to claim 4.

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