KR101938559B1 - Primers for LAMP based detection of Staphylococcus and its use - Google Patents

Abstract

The present invention discloses a primer set used for isothermal amplification that accurately and rapidly detects four Staphylococci causing serious diseases in poultry, and a method for detecting the bacteria using the same. The primer according to the present invention can be widely used in the field of veterinary, food industry, environment and human body because it can confirm the infection or contamination of the pathogen in the infectious source or pollutant as well as high sensitivity and specificity, The detection is possible without any additional processing such as post-electrophoresis, and the convenience is enhanced.

Description

[0001] Primers for detecting staphylococci using LAMP and their uses [0002] Primers for LAMP based detection of Staphylococcus and its use [

The present invention relates to a primer for accurately and rapidly detecting Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus and Staphylococcus xylosus using the LAMP method and its use.

Staphylococcus is a bacterium widely infected by various livestock, including livestock, livestock products, food, and humans. It is infected mainly by S. aureus , but infection and contamination by other species often occur.

Clinical symptoms due to staphylococcal infection in poultry vary according to the infection, and the most frequent infections are bone, tendon sheath, and joint. In addition, it causes infection in skin, sternal bursa, yolk sac, heart, spine, eyelid, testis, When bone and joints are infected, osteomyelitis and arthritis are caused to cause lameness. In case of infection with egg yolk, it causes severe necrosis upon vaginal infection, vascular necrosis, and necrotizing dermatitis when infected with skin, Infection causes chicken fever inflammation or bumblefoot, leading to wilting and causing economic loss in poultry farms. Staphylococcus aureus is known a variety of samples, are separated in a variety of environments, including S. aureus S. xylosus, S. cohnii, S. lentus, S. epidermidis, S. saprophyticus, S. sciuri and S. gallinarum and the like. It is important to identify the exact cause of the source or source of contamination from these various types of samples and environment for the preparation and prevention of post-treatment. As a method for identifying staphylococcal infection or contamination, culture using a selective medium, biochemical characterization or genetic characterization will be used for final identification. These series of methods require the use of media, biochemical test equipment, reagents, equipment and reagents required for genetic testing, and are time-consuming. Therefore, in order to overcome the limitations of the existing diagnostic methods for the development of diagnostic methods that have higher specificity and higher detection sensitivity than the existing diagnostic methods and can increase the application efficiency in the field by using the LAMP method, And it is necessary to develop a detection method with high application efficiency in the field.

U.S. Published Patent Application No. 2015-0232919

The present invention provides a method for detecting the infection of Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus and Staphylococcus xylosus , while exhibiting high sensitivity and specificity.

In one embodiment, the invention provides a primer set for LAMP analysis for detection of Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus, and Staphylococcus xylosus , including F3 primer, B3 primer, FIP primer, BIP primer: SEQ ID NOS: 19-22, A Staphylococcus aureus specific primer set comprising the primers represented by SEQ ID NOS: 31 to 34 or SEQ ID NOS: 37 to 40; A Staphylococcus cohnii specific primer set comprising the primers represented by SEQ ID NOs: 43 to 46, SEQ ID NOs: 49 to 52, SEQ ID NOs: 55 to 58, SEQ ID NOs: 61 to 64, or SEQ ID NOs: 73 to 76; A Staphylococcus lentus comprising the primers represented by SEQ ID NOs: 79 to 82, SEQ ID NOs: 85 to 88, SEQ ID NOs: 91 to 94, SEQ ID NOs: 97 to 100 or SEQ ID NOs: A specific primer set; Staphylococcus xylosus containing the primers represented by SEQ ID NOs: 115 to 118, SEQ ID NOs: 121 to 124 or SEQ ID NOs: 127 to 130 Specific primer set.

The set of primers according to the present invention may further comprise an LF primer and an LB primer for rapid reaction, wherein the Staphylococcus aureus specific primer set is represented by SEQ ID NOs: 23 and 24, SEQ ID NOs: 35 and 36 or SEQ ID NOs: 41 and 42 Primer; Wherein said Staphylococcus cohnii specific primer set comprises a primer set forth in SEQ ID NOs: 47 and 48, SEQ ID NOs: 53 and 54, SEQ ID NOs: 59 and 60, SEQ ID NOs: 65 and 66, or SEQ ID NOs: 77 and 78; The Staphylococcus lentus Specific primer sets include primers represented by SEQ ID NOs: 83 and 84, SEQ ID NOs: 89 and 90, SEQ ID NOs: 95 and 96, SEQ ID NOs: 101 and 102 or SEQ ID NOs: 107 and 108; The Staphylococcus xylosus specific primer set is selected from the primers set forth in SEQ ID NOs: 119 and 120, SEQ ID NOs: 125 and 126 or SEQ ID NOs: 131 and 132, respectively.

When a primer set according to the present invention is required for real time or detection of an amplification product at the termination of the reaction, one or more primers contained in each set may be labeled with a detectable labeling substance.

In another aspect, the disclosure is also directed to a method of screening for Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus , wherein the method comprises detecting Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And providing a sample of a subject suspected of having an infection of Staphylococcus xylosus ; Providing any one of the primer sets according to the present invention; Performing a LAMP reaction using the sample and the primer set; And the results of the LAMP reaction were analyzed and compared with the control samples, the results were compared with those of Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus infection or contamination.

The primer set according to the present invention may be selected from the group consisting of Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus , such as pathogenic emotional samples in the veterinary field, skeletal disease entities in poultry farms, contaminated individuals in slaughter products, mastitis infections in livestock including cattle, milk of infected individuals, But are not limited to, various specimens including skin infections in hospitals or patients with systemic infection, various food and environmental samples.

In the method according to the present invention, the analysis of the LAMP reaction results is possible both in real time or after the termination of the reaction, and the amplification is determined, for example, by measurement using one or more of color change measurement or turbidity, When the color changes or the turbidity increases, it can be judged that the amplification is positive for Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus and Staphylococcus xylosus .

Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus can be specifically detected by an isothermal amplification method, and a method of detecting the bacterium using the primer set can detect contamination of a pathogen rapidly in various specimens as well as high sensitivity and specificity. It can be widely used in veterinary field, food industry field, environment and human body field, and if necessary, detection can be performed without any additional treatment such as electrophoresis after completion of the reaction, thereby enhancing convenience.

FIG. 1 shows the results of detection of the specificity and sensitivity of the combination of Staphylococcus aureus- specific LAMP primers set forth in Table 1 according to one embodiment of the present invention by color change and agarose gel electrophoresis.
FIG. 2 shows the results of detection of the specificity and sensitivity of the combination of Staphylococcus cohnii- specific LAMP primers set forth in Table 2 according to another embodiment of the present invention by color change and agarose gel electrophoresis.
FIG. 3 shows the results of detection of the specificity and sensitivity of the combination of Staphylococcus lentus- specific LAMP primers set forth in Table 3 according to another embodiment of the present invention by color change and agarose gel electrophoresis.
FIG. 4 shows the results of detection of the specificity and sensitivity of the combination of Staphylococcus xylosus- specific LAMP primers set forth in Table 4 according to another embodiment of the present invention by color change and agarose gel electrophoresis.
Sensitivity is shown in red in each of the above figures. For each set, reactions were performed in a total of 7 different concentrations and negative controls, each concentration being as follows; 1, 1: 100 pg /,, 2: 20 pg /,, 3: 4 pg /,, 4: 800 fg /,, 5: 160 fg /,, 6:32 fg / 8: negative control; 7 primer set was 1: 50 pg / μl, 2: 10 pg / μl, 3: 2 pg / μl, 4: 400 fg / μl, 5:80 fg / Negative control; 2, 3, and 4 are graphs showing the relationship between the concentration of 1: 250 pg / μl, 2:50 pg / μl, 3:10 pg / Mu] l and 8: negative control.

We have used Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus can be easily detected with high accuracy and sensitivity at the nucleic acid level.

Staphylococcus is a bacterium widely infected by various livestock, including livestock, livestock products, food, and humans. It is infected mainly by S. aureus , but infection and contamination by other species often occur. The LAMP method was developed to detect the exact cause of the infectious source or contamination source from various kinds of samples and the environment. This LAMP-Staphylo method was developed to detect S. aureus , S. cohnii , S. lentus and S. xylosus , the major isolates of Staphylococcus aureus. The LAMP-Staphylo method was used to prepare four primers for Staphylococcus aureus and to demonstrate a higher sensitivity and specificity than the conventional detection method using Loop-Mediated Isothermal Amplification (LAMP) Can be detected simultaneously.

Thus, in one embodiment, the present invention provides a method of treating Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus A set of specifically detectable primers for detection, and a method for screening for the presence of Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And a method for detecting Staphylococcus xylosus .

In one embodiment, the invention is directed to a method of treating Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And a primer set for LAMP analysis capable of specifically detecting Staphylococcus xylosus .

As used herein, the term " primer " refers to a nucleic acid molecule in which a nucleotide is extended by covalent bonding at its 3 'end in a nucleic acid amplification or synthesis reaction using a polymerase as a single strand of oligonucleotide. The primer set according to the present invention is used for amplification of nucleic acid, i.e., LAMP analysis.

As used herein, the term " nucleic acid " refers to a single or double stranded oligonucleotide or polynucleotide, which refers to an RNA or DNA molecule or analogs and derivatives thereof comprising one or more nucleotides. Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus The nucleic acid may comprise all or a portion of the genome, and in one embodiment, DNA or RNA.

As used herein, the terms " oligonucleotide " and " polynucleotide " are used interchangeably, and both encompass both. The term " primer " can also be used in combination with " oligonucleotide " and " polynucleotide ", including nucleic acid (RNA or DNA), aptamer and the like.

The term " detection " is used herein to refer to the detection of Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus are analyzed for the presence or absence of nucleic acid, or the amount thereof, in particular, based on the LAMP assay described below.

As used herein, the term " target " or " target nucleic acid " refers to a nucleic acid sequence which is specifically amplified by a set of primers according to the present invention, wherein the primer set herein is combined and comprises RNA or DNA.

The primer according to the present invention specifically binds to the target nucleic acid through complementation and amplifies the target nucleic acid by the LAMP method. In the present invention, specifically, only the genes specific to each strain described in Tables 1 to 4 can be specifically detected.

LAMP was amplified by strand amplification using a nucleic acid amplification method (Notomi, T. et al., 2000. Loop-Mediated Isothermal Amplification of DNA, Nucleic Acids Res 28, E63) capable of amplifying a target nucleic acid with high sensitivity and specificity under isothermal conditions and B3 region, B2 region, B1 region, F1c region, F2c region, and F3c region in the 5 '→ 3' direction (the complementary region to the above region is B3c , At least four to six primer sets specifically designated in SEQ ID NOs: 1, 2, 3, 4, 5, isothermal amplification using loop primers, see Mol Cell Probes 16, 223-9). Standard LAMP reactions include at least four of two forward primers FIP, BIP (backward inner primer) and two outer primers F3 (forward outer primer) and B3 (backward outer primer). For accelerated LAMP, a total of six primers can be used, including LF (Loop F) and LB (Loop B).

The LAMP reaction largely involves an initiation structure production step, a cycling amplification step and an elongation and recycling step. In the initiation step, the F2 portion constituting the inner primer FIP binds to the F2c region of the target nucleic acid to initiate the reaction. The outer primer F3 primer then binds to the F3c region of the target nucleic acid to initiate strand replacement DNA synthesis, resulting in single stranded nucleic acid. This single strand forms a loop at the 5 'end through a F1 / F1c junction in which a second internal primer BIP and an external primer B3 bind to result in DNA synthesis and strand replacement DNA synthesis. As a result, a single strand having a stem-loop formed at both ends of the dumbbell shape is formed, and DNA synthesis starts from the 3 'end of the F1 region. In the subsequent cycling amplification step, only two inner primers are used. At first, one inner primer binds to the loop, and strand replacement DNA synthesis occurs. As a result, the original loop-forming single strand and the new loop Stranded single strand is generated, and the stranded DNA synthesis is continued using this as a template, and the amplified product is amplified to about 10 ⁹ within 1 hour. More detailed principles and features and functions of each F3 primer, B3 primer, FIP primer, BIP primer, Loop F primer (LF), and Loop B (LB) primer are described in the aforementioned references Notomi et al., 2000 and Nagamine et al. al., < / RTI > 2002.

As used herein, the term " complementary " or " complementarity " refers to a condition in which a primer or oligonucleotide under sequence hybridization, annealing, or annealing conditions is capable of sequence-specific binding to a target nucleic acid through pairing of Watson- Quot; includes both partial complementarity, substantially complementary and perfectly complementary. Substantially complementarity means that the two strands of the nucleic acid sequence are not completely complementary to each other, but are complementary enough to bind to the target nucleic acid so as to amplify the effect according to the present invention, that is, to amplify the target sequence through the LAMP method.

As used herein, the term " hybridization " or " hybridization " refers to a reaction in which two strands of complementary nucleic acid molecules form a sequence-specific complex through hydrogen bonding. Hybridization can constitute the step of binding the primer to the target nucleic acid or template in the LAMP reaction in which the primer according to the present invention is used. The degree of hybridization is affected by various factors such as the degree of complementation of any two nucleic acid molecules involved, their melting temperature (Tm), or the stringency of hybridization. The hybridization reaction conditions can also be determined in view of this. The stringency of the hybridization reaction is a condition that determines how easily any two nucleic acid molecules that are to be hybridized with each other can be easily conjugated, and the complementary, reaction temperature, ionic strength and / or concentration And can be determined according to the kind, for example, J. Sambrook and DW Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Ed., 2012; and F. M. Ausubel, Ed., Short Protocols in Molecular Biology, Wiley; 5th Ed, 2002, and the like.

Specific binding or hybridization herein means that a particular nucleic acid molecule or primer binds only to the target nucleic acid without substantial binding to a nucleic acid other than the non-target nucleic acid. The primers according to the present invention can specifically bind to the nucleic acids of the target Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus and Staphylococcus xylosus according to the present invention or their complementary sequences under high stringency conditions, and the primer sequence length , Buffer, nucleic acid type, pH, magnesium concentration and reaction temperature.

The target genes for each strain in the present invention are as shown in Tables 1 to 4 and are designed to detect each bacterium with high sensitivity by specifically recognizing each gene region. As described above, in the LAMP reaction, at least four to six primers are designed to detect a target gene, primers and reaction conditions are started to satisfy certain specificity and sensitivity, and one or more strains belonging to the same genus are simultaneously Can be detected.

As described above, at least four types of primers are used in the standard LAMP reaction: FIP (forward inner primer), BIP (backward inner primer), F3 (forward outer primer) and B3 (backward outer primer). For accelerated LAMP, a total of six primers can be used including LF (Loop F) and LB (Loop B). The above mentioned features and functions of each F3 primer, B3 primer, FIP primer, BIP primer, Loop F primer (LF), and Loop B (LB) primer are described in Notomi et al., 2000 and Nagamine et al., 2002 .

A primer specifically binding to a target nucleic acid or a complementary sequence thereof according to the present invention has a length of at least 10 nucleotides, a minimum of 15 nucleotides for F3 and B3, a minimum of 30 nucleotides for FIP and BIP, and a minimum of 30 nucleotides for LF and LB It is 15 nucleotides. With at least 70% complementarity, particularly 80% complementarity, even 90% complementarity, even more particularly 95%, 96%, 97%, 98%, 99% or 100% complementarity with the corresponding portion of the target nucleic acid.

One embodiment The primer according to the present invention comprises substantially the same primer as the primers represented by SEQ ID NOS: 1 to 132, and specifically binds to a target nucleic acid or a complementary sequence thereof. The primers of substantially the same sequence have 70% complementarity, particularly 80% complementarity, more particularly 90% complementarity and even more particularly 95%, 96%, 97%, 98%, 99% or 90% complementarity with the primers shown in SEQ ID NOS: 100% complementarity.

LAMP primers according to the present invention may be selected from the group consisting of target Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus Specifically binds to nucleic acids and detects them with high sensitivity.

Staphylococcus according to the present invention Aureus (Staphylococcus aureus), Staphylococcus Koniyi (Staphylococcus cohnii), Staphylococcus Alkylene Tuscan (Staphylococcus lentus) and Staphylococcus Giles in suspension (Staphylococcus xylosus) LAMP primer set for analysis for staphylococci (Staphylococcus spp.) Detected is selected from the group consisting of is as follows.

The Staphylococcus Aureus For detection The F3 primer is represented by any one of primers selected from the group consisting of SEQ ID NOS: 1, 7, 13, 19, 25, 31 and 37; The B3 primer is represented by any one of primers selected from the group consisting of SEQ ID NOS: 2, 8, 14, 20, 26, 32 and 38; The FIP primer is represented by one primer selected from the group consisting of SEQ ID NOS: 3, 9, 15, 21, 27, 33, or 39; And the BIP primer are represented by any one of a primer selected from the group consisting of SEQ ID NO: 4, 10, 16, 22, 28, 34 or 40, wherein the Staphylococcus The F3 primer for connie detection is represented by any one of primers selected from the group consisting of SEQ ID NOs: 43, 49, 55, 61, 67 or 73 and the B3 primer is represented by SEQ ID NO: 44, 50, 56, 62, 68, 74; and the FIP primer is represented by any one of primers selected from the group consisting of SEQ ID NOS: 45, 51, 57, 63, 69 and 75; The BIP primer is represented by any one of primers selected from the group consisting of SEQ ID NOS: 46, 52, 58, 64, 70, and 76,

The Staphylococcus Rentas The F3 primer for detection is represented by any one of primers selected from the group consisting of SEQ ID NO: 79, 85, 91, 97, or 103; The B3 primer is represented by any one of primers selected from the group consisting of SEQ ID NO: 80, 86, 92, 98, and 104; The FIP primer is represented by any one of primers selected from the group consisting of SEQ ID NOS: 81, 87, 93, 99 and 105; And the BIP primer is represented by any one of primers selected from the group consisting of SEQ ID NO: 82, 88, 94, 100, or 106, and the staphylococcus The F3 primer for xylose detection is represented by one primer selected from the group consisting of SEQ ID NO: 109, 115, 121 or 127; The B3 primer is represented by one primer selected from the group consisting of SEQ ID NO: 110, 116, 122 or 128; The FIP primer is represented by one primer selected from the group consisting of SEQ ID NO: 111, 117, 123 or 129; The BIP primer may be represented by one primer selected from the group consisting of SEQ ID NO: 112, 118, 124, or 130, and may be used in various combinations for each primer of each strain.

Optionally, the primer set may further comprise an LF primer and an LB primer. The stapylloccus The LF primer for detecting aureus is a primer represented by one selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, 29, 35, or 41. The LB primer includes SEQ ID NOs: 6, 12, 18, 24, 30, 36, or 42; The Staphylococcus Konny The LF primer for detection is a primer represented by one selected from the group consisting of SEQ ID NO: 47, 53, 59, 65, 71 or 77 and the LB primer is a primer consisting of SEQ ID NO: 48, 54, 60, 66, 72 or 78 Lt; RTI ID = 0.0 > 1 < / RTI >; The Staphylococcus Rentas The LF primer for detection is a primer represented by one selected from the group consisting of SEQ ID NO: 83, 89, 95, 101 or 107 and the LB primer is selected from the group consisting of SEQ ID NO: 84, 90, 96, Primer to be displayed; And the Staphylococcus Xylose The LF primer for detection is a primer represented by one selected from the group consisting of SEQ ID NO: 113, 119, 125, or 131, and the LB primer is a primer represented by one selected from the group consisting of SEQ ID NO: 114, 120, 126, , And each primer of each strain can be used in various combinations.

In one embodiment, Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And a primer set for LAMP analysis for detection of Staphylococcus xylosus include Staphylococcus aureus comprising the primers shown in SEQ ID NOs: 19 to 22, SEQ ID NOs: 31 to 34 or SEQ ID NOs: 37 to 40 A specific primer set; A Staphylococcus cohnii specific primer set comprising the primers represented by SEQ ID NOs: 43 to 46, SEQ ID NOs: 49 to 52, SEQ ID NOs: 55 to 58, SEQ ID NOs: 61 to 64, or SEQ ID NOs: 73 to 76; A Staphylococcus lentus comprising the primers represented by SEQ ID NOs: 79 to 82, SEQ ID NOs: 85 to 88, SEQ ID NOs: 91 to 94, SEQ ID NOs: 97 to 100 or SEQ ID NOs: A specific primer set; Staphylococcus xylosus containing the primers represented by SEQ ID NOs: 115 to 118, SEQ ID NOs: 121 to 124 or SEQ ID NOs: 127 to 130 Specific primer set.

The set of primers according to the present invention may further comprise an LF primer and an LB primer for rapid reaction, wherein the Staphylococcus aureus specific primer set is represented by SEQ ID NOs: 23 and 24, SEQ ID NOs: 35 and 36 or SEQ ID NOs: 41 and 42 Primer; Wherein said Staphylococcus cohnii specific primer set comprises a primer set forth in SEQ ID NOs: 47 and 48, SEQ ID NOs: 53 and 54, SEQ ID NOs: 59 and 60, SEQ ID NOs: 65 and 66, or SEQ ID NOs: 77 and 78; The Staphylococcus lentus Specific primer sets include primers represented by SEQ ID NOs: 83 and 84, SEQ ID NOs: 89 and 90, SEQ ID NOs: 95 and 96, SEQ ID NOs: 101 and 102 or SEQ ID NOs: 107 and 108; The Staphylococcus xylosus specific primer set is selected from the primers set forth in SEQ ID NOs: 119 and 120, SEQ ID NOs: 125 and 126 or SEQ ID NOs: 131 and 132, respectively.

The primers according to the invention and the methods comprising them can be used for the detection of Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus The present invention can be useful for various types of analysis for detecting contamination or screening for presence or absence of contamination in various samples suspected of infection or contamination,

When a primer set according to the present invention is required for real time or detection of an amplification product at the termination of the reaction, one or more primers contained in each set may be labeled with a detectable labeling substance.

In another aspect, the disclosure is also directed to a method of screening for Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus , wherein the method comprises detecting Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And providing a sample of a subject suspected of having an infection of Staphylococcus xylosus ; Providing any one of the primer sets according to the present invention; Performing a LAMP reaction using the sample and the primer set; And the results of the LAMP reaction were analyzed and compared with the control samples, the results were compared with those of Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus infection or contamination.

The primers according to the invention and the methods comprising them can be used for the detection of Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus The present invention can be useful for various types of analysis for detecting contamination or screening for presence or absence of contamination in various samples suspected of infection or contamination,

Examples of the sample in which the primer set and method according to the present invention can be used include, for example, pathological emotion samples of livestock including poultry, slaughtered products at slaughterhouses, various livestock products such as milk and livestock water; Various foods including drinking water, preserved food, intake food, and foodstuff; feed; Blood, urine, feces, or body fluids; Or environmental samples including water, ground water, water in public facilities, cooking utensils including chopping boards, knives, kitchen cloths, or various equipment or devices used in hospitals.

These biological samples may also be those obtained directly by the usual method of obtaining the samples immediately before the test or stored separately in advance.

In another embodiment, the sample according to the present invention can be used as a sample in which a nucleic acid separated from the above-mentioned sample is used. Separation is separation from the sample containing the nucleic acid, including separation into various purity.

The primers according to the present invention and the methods comprising them are used for LAMP-type analysis for the detection of bacteria in any sample in which there is a possibility that the bacteria are present.

In one embodiment, the reactants comprise 0.2 uM of each F3 and B3 primer, 1.6 uM of each FIP and BIP primer, 0.8 uM of each LF and LB primer, 0.4 M betaine, 10 mM MgSO4, 1.4 mM dNTPs, × ThermoPol reaction buffer (Mmonitor), 8 U Bst DNA polymerase (Mmonitor), and 1.0 to 9.5 μl of sample required analysis.

The reaction is then allowed to react at a temperature suitable for DNA polymerase activity. The reaction temperature can be determined without difficulty in view of the nucleotide sequence of the target enzyme and the enzyme used in the reaction. The reaction is then allowed to react at a time appropriate for amplification of the target nucleic acid.

The amplification reaction product after amplification can be detected in various ways, for example, by color change of reaction solution, turbidity change due to DNA synthesis, fluorescence and / or electrophoresis, and the detected product is positive and / or Is compared with the product of the negative control sample and used for quantitative or qualitative analysis.

In addition, when it is necessary to detect non-specific amplification by non-specific priming of a primer, a non-specific nucleic acid binding agent such as acidium bromide or picogreen is used in a control reaction not containing a template to generate nonspecific total amplification The amount of product can be determined.

The primer set according to the present invention can be usefully used in various analyzes for detecting contamination or screening for the presence or absence of infection in various samples suspected of being infected or contaminated for detection of infection or contamination.

The primers according to the present invention can be used to transform Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus , and Staphylococcus xylosus are detected at the nucleic acid level and compared with the control group or the reference group, depending on the presence or absence of the nucleic acid, And Staphylococcus xylosus infection, contamination, or antibiotic treatment.

Hereinafter, the present invention will be described in detail with reference to the following examples, which are intended to be illustrative and not limit the scope of the present invention in any way.

The present invention may be practiced using conventional techniques within the skill of those skilled in the art of cell biology, cell culture, molecular biology, gene transformation techniques, microbiology, DNA recombinant techniques, unless otherwise indicated. Further, a more detailed description of common techniques can be found in Molecular Biotechnology: (Bernard et al., ASM press 2014); Molecular Cloning: A Laboratory Manual, 4th Ed. (Sambrook et al., Cold Spring Harbor Laboratory Press 2012); Short Protocols in Molecular Biology, 5th Ed. (Ausubel F. et al. Eds., John Wiley & Sons 2002).

Example 1. Staphylococcus  securing the standard strain and preparing the specimen

Staphylococcus aureus (ATCC 25923), Staphylococcus cohnii (ATCC 35662), Staphylococcus lentus (ATCC 49574) And Staphylococcus xylosus (ATCC 29971) were obtained from ATCC (American Type Culture Collection) and Korean Microorganism Preservation Center, and then DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer's method.

Example 2. Staphylococcus Primer-specific primers

Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus , various genes were obtained from NCBI and GenBank, and a final primer specific for the strain was selected. Specifically, the enzyme nuclease (DQ507382), which cleaves the phosphodiester bond present in the nucleic acid as a target of Staphylococcus aureus , and the GTP-binding protein (GenBank accession number, NC_007795), which contributes to the cell viability and stabilization of the ribosome, Staphylococcus cohnii RNA polymerase sigma factor (GenBank accession number, AY234840), a protein essential for the binding of RNA polymerase to the promoter for RNA synthesis, and enzyme catalase (GenBank accession number, EU259827 ), Autolysin (GenBank accession number, AJ310356), which degrades bacterial cell walls for cell division and growth, and ABC transporter permease, which transports C4-dicarboxylate into cells through hydrolysis of C4-dicarboxylate by-products of the GenBank accession number, AB934909), metabolic targets of Staphylococcus lentus The enzyme catalase (GenBank accession number, EU259844) needed to prevent cell damage caused by hydrogen peroxide generated, the enzyme Fructose 1,6-bisphosphatase (GenBank accession number, KF049005) required in the process of gluconeogenesis to synthesize glucose, Ribosomal RNA adenine methylase (GenBank accession number, HE775264), an enzyme that hydrolyzes S-adenosyl-L-methionine and rRNA, and heme, vitamins 3-methylglutaryl-coenzyme A reductase (GenBank accession number, AB546859) required for the Mevalonate pathway, which produces molecules such as K, CoQ10 and D-xylulose 5-phosphate in D-xylulose as a target of Staphylococcus xylosus (GenBank accession number, X57599 S47084), catalase (GenBank accession number, AY687336) and lipid hydrolysis A primer set for each of primers F3, B3, FIP, BIP, LF and LB was prepared based on the lipase (GenBank accession number, AF208229) gene of Doha (Table 1 to Table 4). Each primer sequence was verified using NCBI (National Center for Biotechnology Information), Genbank, and Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi website). As a result, Did not exist. Each primer set was designed and combined to be optimized for the LAMP reaction and showed excellent effects in terms of specificity and sensitivity.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

Example 3. Specificity and Sensitivity of LAMP Reaction

Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus strains. The specificity was confirmed by the color change of the reactants and the electrophoresis results (FIG. 1A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 3A, FIG. 3B, , Figures 3d, 3e, 4a and 4b). Obtained in Example 1. To this end, Staphylococcus aureus, Staphylococcus cohnii, Staphylococcus lentus And Staphylococcus xylosus and 19 other strains were extracted and each DNA was prepared at 25 ng / ul. For LAMP analysis, 0.2 μM of each F3 and B3 primer, 1.6 μM of each FIP and BIP primer, 0.8 μM of each LF and LB primer in a total volume of 25 μl using a Mmiso DNA amplification kit (Mmonitor), 0.4 M betaine , 10 mM MgSO ₄ , 1.4 mM dNTPs, 1 × ThermoPol reaction buffer (Mmonitor), 8 U Bst DNA polymerase (Mmonitor) and 1 μl of the separated DNA was used. Isothermal amplification was performed at 63 ° C for 35-40 minutes in the above composition, followed by reaction at 80 ° C for 5 minutes to terminate the reaction.

The results show that the primer set according to the present invention as set forth in Figures 1a, 1b, 2a, 2b, 2c, 2d, 3a, 3b, 3c, 3d, 3e, 4a and 4b No other similar strains cross-reacted, indicating that only the strains were specifically detected. The above results can sufficiently show the specificity with other combinations of primer sets (Tables 1 to 4).

Next, the primer set of each combination (Table 1 to Table 4), Staphylococcus aureus , Staphylococcus cohnii , Staphylococcus lentus And Staphylococcus xylosus . For this purpose, the prepared Staphylococcus aureus genome was prepared at a concentration of 100 pg, 20 pg, 4 pg, 800 fg, 160 fg, 32 fg and 6 fg in the reaction solution in response to the nuc gene, and 50 pg, 10 pg, Amplification was carried out at 63 ° C. for 35 minutes at the same composition using 2 pg, 400 fg, 80 fg, 16 fg and 3.2 fg, followed by reaction at 80 ° C. for 5 minutes. Sensitivity of the reaction was checked by color change and electrophoresis 1c). Staphylococcus cohnii , Staphylococcus lentus and Staphylococcus xylosus Isothermal amplification was carried out at 63 ° C for 35 min in the same composition using 250 μg, 50 pg, 10 pg, 2 pg, 400 fg, 80 fg and 16 fg of the dielectrics in the reaction solution, followed by reaction at 80 ° C. for 5 minutes. And the sensitivity was confirmed as a result of electrophoresis (FIGS. 2E, 3F and 4C).

Therefore, before the reaction, it is present in purple, and as the DNA synthesis reaction occurs, the concentration of Mg ²⁺ ions decreases and thus the color changes from purple to blue. A change in color is a positive reaction indicating that the nucleic acid is amplified.

The method according to the present invention is advantageous in qualitative and quantitative analysis when absorbance is measured at a wavelength of 650 nm using a spectrophotometer.

Detection of bacteria using the primer set according to the present invention, as described in Figures 1c, 2e, 3f and 4c, may be performed at a concentration of 2 pg to 400 fg when the sensitivity is based on the genomic DNA of Staphylococcus aureus ; 2 pg to 400 fg when using the genomic DNA of Staphylococcus cohnii ; 2pg to 80fg using the genomic DNA of Staphylococcus lentus ; The genomic DNA of Staphylococcus xylosus was found to be as high as 50 pg to 80 fg.

<110> REPUBLIC OF KOREA (Animal and Plant Quarantine Agency) <120> Primers for LAMP based detection of Staphylococcus and its use <130> DP201704007P <160> 132 <170> KoPatentin 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > F3 primer for LAMP detection of Staphylococcus aureus nuc <400> 1 aaattacata aagaacctgc gac 23 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > B3 primer for LAMP detection of Staphylococcus aureus nuc <400> 2 tgtcaaactc gacttcaatt ttc 23 <210> 3 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus aureus nuc <400> 3 ggtgtatcaa ccaataatag tcttaaagcg attgatggtg atacgg 46 <210> 4 <211> 48 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BIP primer for LAMP detection of Staphylococcus aureus nuc <400> 4 caaagcatcc taaaaaaggt gtagttgcat tttctaccat ttttttcg 48 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus aureus nuc <400> 5 atgtcattgg ttgacctttg tacatta 27 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus aureus nuc <400> 6 aatatggtcc tgaagcaagt gc 22 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > F3 primer for LAMP detection of Staphylococcus aureus nuc <400> 7 gttgtagttt caagtctaag tagc 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > B3 primer for LAMP detection of Staphylococcus aureus nuc <400> 8 caggtgtatc aaccaataat agtc 24 <210> 9 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus aureus nuc <400> 9 gaagttgcac tatatactgt tggatcagca aatgcatcac aaacag 46 <210> 10 <211> 46 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BIP primer for LAMP detection of Staphylococcus aureus nuc <400> 10 aattacataa agaacctgcg actgtcattg gttgaccttt gtacat 46 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus aureus nuc <400> 11 ttcagaacca cttctattta cgcc 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus aureus nuc <400> 12 ttaaagcgat tgatggtgat acgg 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > F3 primer for LAMP detection of Staphylococcus aureus nuc <400> 13 gactattatt ggttgataca cctg 24 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > B3 primer for LAMP detection of Staphylococcus aureus nuc <400> 14 cgaactaaag cttcgtttac c 21 <210> 15 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus aureus nuc <400> 15 ctttgcattt tctaccattt ttttcgaagc atcctaaaaa aggtgtaga 49 <210> 16 <211> 44 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BIP primer for LAMP detection of Staphylococcus aureus nuc <400> 16 attgaagtcg agtttgacaa agtccatcag cataaatata cgct 44 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus aureus nuc <400> 17 gcacttgctt caggaccata tttc 24 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus aureus nuc <400> 18 caaagaactg ataaatatgg acgtggc 27 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > F3 primer for LAMP detection of Staphylococcus aureus nuc <400> 19 ccaacagtat atagtgcaac ttc 23 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > B3 primer for LAMP detection of Staphylococcus aureus nuc <400> 20 ttgcattttc taccattttt ttcg 24 <210> 21 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus aureus nuc <400> 21 aatgtcattg gttgaccttt gtacaattac ataaagaacc tgcgac 46 <210> 22 <211> 46 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BIP primer for LAMP detection of Staphylococcus aureus nuc <400> 22 gactattatt ggttgataca cctgacactt gcttcaggac catatt 46 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus aureus nuc <400> 23 aaccgtatca ccatcaatcg c 21 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus aureus nuc <400> 24 caaagcatcc taaaaaaggt gtagaga 27 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus aureus engA <400> 25 acttgattta cctacgtttg gt 22 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Staphylococcus aureus engA <400> 26 aaatgcgtac agacgtgtat 20 <210> 27 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus aureus engA <400> 27 gttagatgca gttgtttctc atttcgtcca ataatggata gtcgaattg 49 <210> 28 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Staphylococcus aureus engA <400> 28 aagtcaccaa gacctaaacc atgtttctat tcattaggat ttggt 45 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus aureus engA <400> 29 tggtgaagag gaagaagatc cttatga 27 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus aureus engA <400> 30 tgaccctgat atcggatacg gt 22 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus aureus engA <400> 31 gcttcatcta tggcgattt 19 <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Staphylococcus aureus engA <400> 32 ttaatagaat agttggagaa cgtg 24 <210> 33 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus aureus engA <400> 33 ttcaatatta ttgatacagg tggtactgcc tgcgctctaa tttgtgttt 49 <210> 34 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Staphylococcus aureus engA <400> 34 catgtgttaa ccattcacct gaagtttcga ttgtggaaga cacg 44 <210> 35 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus aureus engA <400> 35 tgaaattggt gatgcaccat tcc 23 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus aureus engA <400> 36 tacgatctcg tgttacacct gg 22 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus aureus engA <400> 37 tgtatcaata gcgtctctcg t 21 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Staphylococcus aureus engA <400> 38 cacatggttt aggtcttggt 20 <210> 39 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus aureus engA <400> 39 accaaacgta ggtaaatcaa gtttccctgc aacattagaa acgata 46 <210> 40 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Staphylococcus aureus engA <400> 40 cgtccaataa tggatagtcg aattgacttg ttagatgcag ttgtttctc 49 <210> 41 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus aureus engA <400> 41 tgctatttta ggtgaagatc gcgt 24 <210> 42 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus aureus engA <400> 42 cataaggatc ttcttcctct tcaccaa 27 <210> 43 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus cohnii RNA          폴리머마 시마 계수 <400> 43 gagaaaatga agtttcggtt tca 23 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Staphylococcus cohnii RNA          폴리머마 시마 계수 <400> 44 gcagcatact ttaaaatcct ttca 24 <210> 45 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus cohnii RNA          폴리머마 시마 계수 <400> 45 ttcttccaaa atttcattgg acattgaagg attcaatgag cagaa 45 <210> 46 <211> 48 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BIP primer for LAMP detection of Staphylococcus cohnii RNA          폴리머마 시마 계수 <400> 46 aatcagaagg ggtaacaatc ataaaaagct agcatacaaa gtactctg 48 <210> 47 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus cohnii RNA          폴리머마 시마 계수 <400> 47 ctctatacgc gtccgtggt 19 <210> 48 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus cohnii RNA          폴리머마 시마 계수 <400> 48 aagaagattg tccccttgct ttt 23 <210> 49 <211> 24 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > F3 primer for LAMP detection of Staphylococcus cohnii katA <400> 49 atatgagaag tgcacaaaac aact 24 <210> 50 <211> 18 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > B3 primer for LAMP detection of Staphylococcus cohnii katA <400> 50 gcgttctgtg gtggaatt 18 <210> 51 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus cohnii katA <400> 51 ccttaggaat accacggtct gggatttctg gacatcttta cc 42 <210> 52 <211> 41 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BIP primer for LAMP detection of Staphylococcus cohnii katA <400> 52 gctttagaaa catgcacgga cccaaacacg ttcaccttta t 41 <210> 53 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus cohnii katA <400> 53 aattgttact tggtgcaatg cttca 25 <210> 54 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus cohnii katA <400> 54 ggctcccata cgtattctat gtaca 25 <210> 55 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus cohnii asc <400> 55 gtacatgcct atgtagatgg taat 24 <210> 56 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Staphylococcus cohnii asc <400> 56 caggagcaag tccataatat tgta 24 <210> 57 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus cohnii asc <400> 57 taaagcgatc gtttgcagct ggtatcattg aaacagctaa tacag 45 <210> 58 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Staphylococcus cohnii asc <400> 58 gtagaattag tccatacaca cgacttgtag ctgcatagtc tgcatag 47 <210> 59 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus cohnii asc <400> 59 ccagctcccc atgctaagta at 22 <210> 60 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus cohnii asc <400> 60 gattcatttg cgcgttcaat cact 24 <210> 61 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus cohnii asc <400> 61 gaattagtcc atacacacga ct 22 <210> 62 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Staphylococcus cohnii asc <400> 62 ctaaataacc atggggatcg at 22 <210> 63 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus cohnii asc <400> 63 gtcaggagca agtccataat attggattca tttgcgcgtt caatcac 47 <210> 64 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Staphylococcus cohnii asc <400> 64 gcgctgagta cgatggtgtt ggtctgatcc acctaaataa ttac 44 <210> 65 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus cohnii asc <400> 65 aggtttgtag ctgcatagtc tgc 23 <210> 66 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus cohnii asc <400> 66 ggaactgttt ggacgcataa agc 23 <210> 67 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus cohnii asc <400> 67 tgatacttct aacagctctg aag 23 <210> 68 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Staphylococcus cohnii asc <400> 68 tagaagtctc tgaatcatca tcat 24 <210> 69 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus cohnii asc <400> 69 gtcagagctt acctctgtaa tagacgttaa cactgacgtt actc 44 <210> 70 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Staphylococcus cohnii asc <400> 70 ttcaacttct gatgcagtaa acgtgagtaa cgttagtatt gtcgtc 46 <210> 71 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus cohnii asc <400> 71 ggccgctgaa tcatcatcat t 21 <210> 72 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus cohnii asc <400> 72 catgatgctt ctaacaagac tgaagtc 27 <210> 73 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus cohnii          C4-dicarboxylate ABC transporter permease <400> 73 gagcatatta ccaacgatat tgg 23 <210> 74 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Staphylococcus cohnii          C4-dicarboxylate ABC transporter permease <400> 74 atcacgctgt tattaatttt acca 24 <210> 75 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus cohnii          C4-dicarboxylate ABC transporter permease <400> 75 caacggcggt atagcattta gatgggctaa tgatcctggt aatctac 47 <210> 76 <211> 47 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BIP primer for LAMP detection of Staphylococcus cohnii          C4-dicarboxylate ABC transporter permease <400> 76 gatacccgct agcatcgtaa aatctttgat gtcactatcg taactgc 47 <210> 77 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus cohnii          C4-dicarboxylate ABC transporter permease <400> 77 tagttaactt tgcgaaggtg ctcg 24 <210> 78 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus cohnii          C4-dicarboxylate ABC transporter permease <400> 78 aataccagtt accatacgct gtgc 24 <210> 79 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus lentus katA <400> 79 ttccaaaagg tttccgtaac at 22 <210> 80 <211> 19 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > B3 primer for LAMP detection of Staphylococcus lentus katA <400> 80 ccttcttcga ttgcgttga 19 <210> 81 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus lentus katA <400> 81 tgaagtggaa tttaacccat actcatggtt tcggttctca cactt 45 <210> 82 <211> 48 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BIP primer for LAMP detection of Staphylococcus lentus katA <400> 82 tcaacaaggt attgaaaact acacgtctct ttgacttgat tcacggtc 48 <210> 83 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus lentus katA <400> 83 tttcgccttc atcgttgtac attg 24 <210> 84 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus lentus katA <400> 84 tgaagaagcg gaacaagtta ttgc 24 <210> 85 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus lentus fbp <400> 85 gatgttagtg tcgcgtactt 20 <210> 86 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Staphylococcus lentus fbp <400> 86 cttttcaaaa gcgtaccaat caa 23 <210> 87 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus lentus fbp <400> 87 aagaaaacgt acttctgaac ggacgttcga gttcttcgtc taca 44 <210> 88 <211> 43 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BIP primer for LAMP detection of Staphylococcus lentus fbp <400> 88 tgtgccaaag tgtttatgcg aggtatcgct ggctatacat tac 43 <210> 89 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus lentus fbp <400> 89 cgcagatgaa ttgtctgtca gacg 24 <210> 90 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus lentus fbp <400> 90 ccactagttg catgccaaat gagt 24 <210> 91 <211> 21 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > F3 primer for LAMP detection of Staphylococcus lentus hsp60 <400> 91 cagtaggtat tcgtgaaggt a 21 <210> 92 <211> 23 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > B3 primer for LAMP detection of Staphylococcus lentus hsp60 <400> 92 cttcaattgt gataactcca tcg 23 <210> 93 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus lentus hsp60 <400> 93 gtgcaatttc ttctttcttc tctcgataag gcagttaaag ttgc 44 <210> 94 <211> 45 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BIP primer for LAMP detection of Staphylococcus lentus hsp60 <400> 94 aagtaggttc tatctctgca gttacctact ttttccatag cttct 45 <210> 95 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus lentus hsp60 <400> 95 caggttgtga aatctcgtgt aattctt 27 <210> 96 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus lentus hsp60 <400> 96 ctgacgagga aattggtaaa tttatttc 28 <210> 97 <211> 20 <212> DNA <213> Artificial Sequence <220> F3 primer for LAMP detection of Staphylococcus lentus erm (43) <400> 97 aattgtttct gctggaggaa 20 <210> 98 <211> 22 <212> DNA <213> Artificial Sequence <220> B3 primer for LAMP detection of Staphylococcus lentus erm (43) <400> 98 gtaatcatag cctttcttcc ca 22 <210> 99 <211> 48 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > FIP primer for LAMP detection of Staphylococcus lentus erm (43) <400> 99 ctcaattgga ctaacactcc attgccttga tggatttgat ttaacaat 48 <210> 100 <211> 45 <212> DNA <213> Artificial Sequence <220> BIP primer for LAMP detection of Staphylococcus lentus erm (43) <400> 100 attaacagct tcagccatta tcgatgtatc cgttaattta ccgag 45 <210> 101 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus lentus erm (43) <400> 101 aataatcaat ggcatagcga cagc 24 <210> 102 <211> 21 <212> DNA <213> Artificial Sequence <220> LB primer for LAMP detection of Staphylococcus lentus erm (43) <400> 102 gttccttcat tggtgctgca t 21 <210> 103 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus lentus mva <400> 103 tttgctagtg agtttgaccc 20 <210> 104 <211> 24 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > B3 primer for LAMP detection of Staphylococcus lentus mva <400> 104 catctgttgg atatactggc ttaa 24 <210> 105 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus lentus mva <400> 105 agcgtatgaa tacctgatgc tgcaatacat gcatttagat catga 45 <210> 106 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Staphylococcus lentus mva <400> 106 cgctttatca ttcaaattat gggtttccat cttagtcccg gcaatc 46 <210> 107 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus lentus mva <400> 107 aacaccttga aacatacctt gactagc 27 <210> 108 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus lentus mva <400> 108 gaagagggta agtatggtga tgatg 25 <210> 109 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus xylosus xylB <400> 109 ctttaaccat tgtgttccaa acc 23 <210> 110 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Staphylococcus xylosus xylB <400> 110 gaaactacct cgcactgaag 20 <210> 111 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus xylosus xylB <400> 111 gtagtgaaat tttcatcagc agagacatta tgggtgtgac tttatctg 48 <210> 112 <211> 46 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BIP primer for LAMP detection of Staphylococcus xylosus xylB <400> 112 agatattaat caatcagaag ttggttatgt ggcgtacgtt caccta 46 <210> 113 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus xylosus xylB <400> 113 taagccattc gaggctatat ccag 24 <210> 114 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus xylosus xylB <400> 114 gctaatggat taatgtacac gcctt 25 <210> 115 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus xylosus katA <400> 115 tcggtaaaca gacagaaatg tt 22 <210> 116 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Staphylococcus xylosus katA <400> 116 ttacgtttaa ccacgtggtt taa 23 <210> 117 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus xylosus katA <400> 117 acttcaaggc aaatcctcta atatgcacga ttttcaactg tagca 45 <210> 118 <211> 45 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BIP primer for LAMP detection of Staphylococcus xylosus katA <400> 118 tacagacgaa ggtaactggg attgggaata atttagggtc tctaa 45 <210> 119 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus xylosus katA <400> 119 gttctgcatc gcctgcac 18 <210> 120 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus xylosus katA <400> 120 gttggtaata atacgcccgt attcttc 27 <210> 121 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus xylosus gehM <400> 121 tggggtggag ataaattgaa tat 23 <210> 122 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Staphylococcus xylosus gehM <400> 122 gttttaccat aacgttcatg acc 23 <210> 123 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus xylosus gehM <400> 123 acgatcgtag ttactactta atgcccgcca agatttagaa agtaatgg 48 <210> 124 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Staphylococcus xylosus gehM <400> 124 gccgttgagc tttattacta tattgtactt ctcagcatgg gctgca 46 <210> 125 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus xylosus gehM <400> 125 accaacgctc gcttcgtatg 20 <210> 126 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus xylosus gehM <400> 126 gggtggcacg gtagattatg g 21 <210> 127 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Staphylococcus xylosus gehM <400> 127 tgcatggttt caatggcttt a 21 <210> 128 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Staphylococcus xylosus gehM <400> 128 ccgtgccacc cttaatatag ta 22 <210> 129 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Staphylococcus xylosus gehM <400> 129 ttctaaatct tggcggatat tcaactgatg atattaatcc agcggta 47 <210> 130 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Staphylococcus xylosus gehM <400> 130 agtaatggtt atgaaacata cgacaacggc acgatcgtag ttac 44 <210> 131 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Staphylococcus xylosus gehM <400> 131 tttatctcca ccccaataat gagc 24 <210> 132 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Staphylococcus xylosus gehM <400> 132 cgagcgttgg tgcattaag 19

Claims (10)

Containing the F3 primer, B3 primer, FIP primer and a BIP primer, Staphylococcus aureus (Staphylococcus aureus), Staphylococcus koniyi (Staphylococcus cohnii), Staphylococcus alkylene Tuscan (Staphylococcus lentus) and Staphylococcus Giles with a switch (Staphylococcus xylosus) primer set for LAMP analysis for Staphylococcus aureus (Staphylococcus spp.) detected is selected from the group consisting of,
A primer set for detecting Staphylococcus aureus comprises a primer set comprising a primer represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; A primer set comprising a primer represented by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10; A primer set comprising a primer represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; A primer set comprising a primer represented by SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22; A primer set comprising a primer represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28; A primer set comprising a primer represented by SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34; Or a primer set comprising a primer represented by SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40;
A primer set for detecting the staphylococcus konyi comprises a primer set comprising a primer represented by SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46; A primer set comprising a primer represented by SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52; A primer set comprising a primer represented by SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58; A primer set comprising a primer represented by SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64; A primer set comprising a primer represented by SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO: 70; Or a primer set comprising a primer represented by SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76;
A primer set for detecting staphylococcus lentus comprises a primer set comprising a primer represented by SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 and SEQ ID NO: 82; A primer set comprising a primer represented by SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87 and SEQ ID NO: 88; A primer set comprising a primer represented by SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93 and SEQ ID NO: 94; A primer set comprising a primer represented by SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100; Or a primer set comprising a primer represented by SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106; And
A primer set for detecting the staphylococcus xylosis comprises a primer set comprising a primer represented by SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111 and SEQ ID NO: 112; A primer set comprising a primer represented by SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 and SEQ ID NO: 118; A primer set comprising a primer represented by SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123 and SEQ ID NO: 124; Or a primer set comprising a primer represented by SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129 and SEQ ID NO: 130.
The method according to claim 1,
A primer set for LAMP analysis for staphylococcal detection, wherein each of the primer sets is composed of six primer sets further comprising LF primer and LB primer as follows:
A primer set for detecting Staphylococcus aureus comprises a primer set comprising a primer represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; A primer set comprising a primer represented by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12; A primer set comprising a primer represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18; A primer set comprising a primer represented by SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24; A primer set comprising a primer represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30; A primer set comprising a primer represented by SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36; Or a set of primers comprising a primer represented by SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42;
A primer set for detecting Staphylococcus konyi comprises a primer set comprising a primer represented by SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48; A primer set comprising a primer represented by SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54; A primer set comprising a primer represented by SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60; A primer set comprising a primer represented by SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66; A primer set comprising a primer represented by SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72; Or a primer set comprising a primer represented by SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78;
A primer set for detecting staphylococcus lentus comprises a primer set comprising a primer represented by SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84; A primer set comprising a primer represented by SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90; A primer set comprising a primer represented by SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96; A primer set comprising a primer represented by SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102; Or a set of primers comprising a primer represented by SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107 and SEQ ID NO: 108; And
A primer set for detecting the staphylococcus xylosis comprises a primer set comprising a primer represented by SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113 and SEQ ID NO: 114; A primer set comprising a primer represented by SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 120; A primer set comprising a primer represented by SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 126; Or a primer set comprising a primer represented by SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132.
delete delete 3. The method according to claim 1 or 2,
A primer set for LAMP analysis for staphylococcal detection, wherein at least one primer contained in each primer set is labeled with a detectable labeling substance.
A method for detecting Staphylococci by the LAMP method in Invitro using a primer set according to claim 1 or 2, the method comprising the steps of:
Providing a sample suspected of having staphylococcal infection or contamination;
Providing a primer set according to claim 1 or 2;
Performing a LAMP reaction using the sample and the primer set; And
Analyzing the result of the reaction during or after the LAMP reaction and comparing the result to a control sample, if there is a difference, determining the sample as a staphylococcal infection or contamination.
The method according to claim 6,
The sample may be a sample of a diseased animal including poultry, a slaughtered product at a slaughterhouse, various livestock products and livestock water, a milk test sample, a blood test sample, a feces, a urine or a body fluid, a food, a food, Drinking water, or water in a public facility, a cooking utensil, or a hospital apparatus.
The method according to claim 6,
Wherein the analysis of the LAMP reaction result is determined by measurement using at least one of a color change measurement of the reaction result or turbidity.
A composition for detecting staphylococci using the LAMP method comprising the primer set according to any one of claims 1 to 3.
A kit for detecting staphylococci using the LAMP method, comprising the primer set according to claim 1 or 2.

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