WO2020101194A1 - Primer set for loop-mediated isothermal amplification reaction for diagnosing carbapenemase-producing enterobacteriaceae, and use thereof - Google Patents

Abstract

The present invention pertains to: a primer set for loop-mediated isothermal amplification reaction for diagnosing carbapenemase-producing enterobacteriaceae; and a use thereof. More specifically, the present invention pertains to: a primer set for loop-mediated isothermal amplification reaction for IMP, VIM, KPC, and NDM, which are known as carbapenemase-producing genes; and a method for diagnosing carbapenemase-producing enterobacteriaceae using same. According to the present invention, it was found that a primer set for loop-mediated isothermal amplification reaction for detecting carbapenemase-producing enterobacteriaceae can be used to perform a test within one hour using only simple equipment, and effectively detect only carbapenemase-producing enterobacteriaceae, and can thus effectively provide information on whether to administer carbapenem.

Description

Set of primers for loop-mediated isothermal amplification for the diagnosis of carbapenemase-producing intestinal bacteria and uses thereof

The present invention relates to a primer set for loop-mediated isothermal amplification reaction for the diagnosis of carbapenemase-producing intestinal bacteria and uses thereof, and more specifically, loop-mediated isotherm for IMP, VIM, KPC, and NDM known as carbapenemase-generating genes. A primer set for amplification reaction and a method for diagnosing intestinal bacteria producing carbapenemase using the same.

Carbapenem-based antibiotics are widely used antibiotics for the treatment of Gram-negative bacterial infections. Antibiotics of the carbapenem family include imipenem, meropenem, Invanz, ertapenem, and doripenem. Currently, the carbapenem-based antibiotics are known to be the most powerful antibiotics, and are mainly used for patients with severe infections.

However, due to the indiscriminate use of antibiotics, bacteria that are resistant to various antibiotics occur, which is a social problem. Carbapenem-based antibiotics have also become a major social problem as resistant strains have been reported worldwide. The intestinal bacteria resistant to these carbapenem-resistant antibiotics are called carbapenem resistant enterobacteriaceae (CRE). It is known that intestinal bacteria resistant to these carbapenem-based antibiotics produce an enzyme called β-lactamase to neutralize the carbapenem-based antibiotics, and β-lactamase inactivates antibiotics. (Enzyme inactivation) causing antibiotic resistance. Bacteria that are resistant to the antibiotics of the carbapenem family are E. coli and pneumococcal ( Klebsiella pneumoniae ), as well as several strains that have genes that can produce β-lactamase enzymes. It is known to have.

As genes generating β-lactamase, genes such as KPC, IMP, GES, SIM, and VIM, as well as NDM genes, have been reported. Genes that generate such β-lactamase enzymes exist in several genotypes. The most representative of these, NDM (New Delhi metallo beta lactamase), was named in 2008 after the Swedish patient was confirmed to be infected during surgery in New Delhi, India. Bacteria carrying the NDM gene are known to be resistant to almost all existing antibiotics, and are fatal, and infection experts are concerned because they are likely to spread worldwide in recent years in India and Pakistan, as well as in the United States and Japan. As such, the risk of multi-drug resistant bacteria including CRE has emerged worldwide, and in Korea, in September 2010, 'Cabapenem-resistant enteric bacteria (CRE)' among multi-drug resistant bacteria was urgently designated as a legal infectious disease.

However, recently, a carbapenemase producing enterobacteriaceae (CPE) that produces an enzyme called carbapenemase that can directly degrade carbapenem antibiotics has appeared. In Korea, a total of 59 CPEs were identified by 2012, and in July 2013, as new CPEs that were not reported in Korea were identified, epidemiological investigations were conducted at the Center for Disease Control and confirmed that 63 hospitals and 63 patients were infected with CPE. Became. CPE is a type of CRE having a gene that generates an enzyme capable of directly degrading antibiotics among CREs, and has the potential to transmit resistance to other strains by a plasmid. Representative CPEs include Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase-1 (NDM-1).

In general, the methods of testing for the presence of carbapenem-resistant enterococci are disc diffusion method, Modified Hodge Test (MHT) method, Carbapenemase Nordmann-poirel test, Carbapenemase inhibition test, PCR amplification · Electrophoresis, DNA sequencing methods are used.

Conventional disc diffusion method for the detection of infection with intestinal bacteria resistant to carbapenem is low sensitivity caused by technical errors such as the type of culture medium, dilution ratio, and ambiguous reading of test results, and above all, disadvantages of delayed test results Because of this, there is a limit to quickly and accurately determining the presence or absence of infection with carbapenem-resistant enterococci, and the DNA sequencing test is accurate, but only one sample can be tested with one or two tests. In addition to the high inspection cost, there is a disadvantage that the inspection takes a long time.

In addition, a microarray-type DNA chip is a method that can simultaneously test tens to tens of thousands of genes on a single slide, or an endpoint analysis that simply identifies the genotype by hybridizing the product after the PCR amplification reaction to a DNA chip integrated with a probe. (end-point analysis) method. However, the PCR amplification process is a linear step in which the amount of DNA is arithmeticly increased as the amplification rate begins to decrease as the components required for PCR amplification gradually deplete after exponential phases with a constant DNA amplification rate and high reproducibility. The PCR amplification stops after (linear phases) and proceeds to the plateau phases with the lowest reproducibility. For this reason, the biggest problem of the microarray DNA chip method is that it is difficult to design primers and probes for PCR amplification that can detect multiple target genes while satisfying both specificity and sensitivity, and it is difficult to design a fluorescence intensity cutoff value for positive and negative determination. There is a problem that is difficult to determine.

In order to solve this problem, the specificity and sensitivity of the carbapenem-resistant enterococci are quickly and accurately determined based on the quantified data, and the specificity and sensitivity are high, instead of the conventional disk diffusion method, DNA sequencing method, or multiplex microarray DNA chip. The development of a method that can be done is an urgent situation.

Carbapenemase-producing intestinal bacteria are currently diagnosed or diagnosed within 2 hours using Xpert (Cepheid) using real-time PCR, but in the case of Expert, expensive equipment and reagents are required. The disadvantage is that it takes two hours.

On the other hand, the loop-mediated isothermal amplification (LAMP) method is a simple and fast isothermal amplification method using a Bst DNA polymerase having a strand displacement DNA synthesis function. As a method of continuously amplifying after forming a loop using two pairs of specific primers, specific DNA can be amplified in one step without expensive equipment. It is used as a detection method.

Thus, in the present invention, as a result of efforts to quickly and effectively diagnose intestinal bacteria producing carbapenemase, a primer set for the LAMP reaction was prepared, and the primer set of the present invention was equipped with equipment such as a simple heating block. It was confirmed that the test could be performed within 1 hour, and it was confirmed that the intestinal bacteria producing carbapenemase were effectively diagnosed, and the present invention was completed.

An object of the present invention is to provide a set of primers for loop-mediated isothermal amplification reaction for the diagnosis of carbapenemase-producing intestinal bacteria.

Another object of the present invention is to provide a carbapenemase-producing enterococcal bacteria diagnostic kit comprising the primer set for the loop-mediated isothermal amplification reaction.

Another object of the present invention is to provide a method for diagnosing intestinal bacteria producing carbapenemase using the primer set for the loop-mediated isothermal amplification reaction.

In order to solve the above problems, the present invention

(1) Primer set for IMP gene detection consisting of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 6;

(2) a primer set for VIM gene detection consisting of the nucleotide sequence of SEQ ID NO: 15 to SEQ ID NO: 20;

(3) KPC gene detection primer set consisting of the nucleotide sequence of SEQ ID NO: 29 to SEQ ID NO: 34; And

(4) NDM gene detection primer set consisting of the nucleotide sequence of SEQ ID NO: 43 to SEQ ID NO: 48; Provided is a primer set for loop-mediated isothermal amplification (LAMP) for detecting carbapenemase producing enterobacteriaceae (CPE), which is composed of any one or more primer sets.

In a preferred embodiment of the present invention, the primer set for IMP gene detection is a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, a forward inner primer of SEQ ID NO: 3 (FIP), SEQ ID NO: 4 It consists of a reverse inner primer (Back inner pimer; BIP), a forward loop primer of SEQ ID NO: 5 (LoopF) and a reverse loop primer of SEQ ID NO: 6 (LoopB),

The primer set for VIM gene detection includes a forward primer of SEQ ID NO: 15, a reverse primer of SEQ ID NO: 16, a forward inner primer of SEQ ID NO: 17 (FIP), and a reverse inner primer of SEQ ID NO: 18 (Back inner pimer; BIP) ), A forward loop primer of SEQ ID NO: 19 (LoopF) and a reverse loop primer of SEQ ID NO: 20 (LoopB),

The KPC gene detection primer set includes a forward primer of SEQ ID NO: 29, a reverse primer of SEQ ID NO: 30, a forward inner primer of SEQ ID NO: 31 (FIP), and a reverse inner primer of SEQ ID NO: 32 (Back inner pimer; BIP) ), A forward loop primer of SEQ ID NO: 33 (LoopF) and a reverse loop primer of SEQ ID NO: 34 (LoopB),

The NDM gene detection primer set includes a forward primer of SEQ ID NO: 43, a reverse primer of SEQ ID NO: 44, a forward inner primer of SEQ ID NO: 45 (FIP), and a reverse inner primer of SEQ ID NO: 46 (Back inner pimer; BIP). ), The forward loop primer of SEQ ID NO: 47 (LoopF) and the reverse loop primer of SEQ ID NO: 48 (LoopB).

In another preferred embodiment of the present invention, the carbapenemase-producing enterococcus is Escherichia coli , Klebsiella pneumonia , Pseudomonas spp. , Serratia spp ., Citro Citrobacter spp. , Acinetobacter spp. , It may be one or more selected from the group consisting of Morganella spp. , Providencia spp. And Enterobacter spp .

The present invention also provides a kit for detecting carbapenemase-producing intestinal bacteria comprising a primer set for loop-mediated isothermal amplification (LAMP).

In a preferred embodiment of the present invention, the kit may further include a DNA polymerase for loop-mediated isothermal amplification reaction, dNTP (dATP, dCTP, dGTP and dTTP) mixture, buffer solution, and distilled water.

The present invention also

(A) separating the DNA from the sample;

(b) amplifying a target sequence by performing the loop-mediated isothermal amplification reaction using the extracted DNA as a template and using the primer set; And

(c) detecting the amplification product, it provides a method for detecting intestinal bacteria producing carbapenemase.

In a preferred embodiment of the present invention, the sample of step (a) may be obtained by feces or rectal swab.

In another preferred embodiment of the present invention, the loop-mediated isothermal amplification reaction may be performed at 60 ° C to 70 ° C.

The present invention confirmed that the primer set for loop-mediated isothermal amplification reaction for the detection of carbapenemase-producing intestinal bacteria can be carried out within 1 hour with simple equipment, effectively detecting carbapenemase-producing intestinal bacteria. Since it was confirmed, it is possible to effectively provide information on whether or not to administer carbapenem.

1 is a schematic diagram of a loop-mediated isothermal amplification (LAMP) method.

FIG. 2 is data comparing the sensitivity of detecting carbapenemase-producing gut bacteria of two types of IMP gene loop-mediated isothermal amplification reactions with general PCR and real-time PCR. 2A to 2C are results of detecting intestinal bacteria producing carbapenemase according to each sample sample. IMP-1 refers to the primer set in Table 1, IMP-2 refers to the primer set in Table 2, the curve marked with ○ indicates a positive control, and the curve marked with × indicates a negative control.

FIG. 3 is data comparing the sensitivity of detecting carbapenemase-producing gut bacteria of two types of VIM gene loop-mediated isothermal amplification reactions with general PCR and real-time PCR. 3A to 3F are results of detecting intestinal bacteria producing carbapenemase according to each sample sample. VIM-1 refers to the primer set in Table 3, VIM-2 refers to the primer set in Table 4, the curve marked with ○ indicates a positive control, and the curve marked with X indicates a negative control.

FIG. 4 is data comparing the sensitivity of detecting carbapenemase-producing intestinal bacteria of two types of KPC gene loop-mediated isothermal amplification reactions with general PCR and real-time PCR. 4A to 4E are results of detecting intestinal bacteria producing carbapenemase according to each sample sample. KPC-1 refers to the primer set in Table 5, KPC-2 refers to the primer set in Table 6, the curve marked with o indicates a positive control, and the curve marked with x indicates a negative control.

FIG. 5 is data comparing the sensitivity of detecting carbapenemase-producing intestinal bacteria of two types of NDM gene loop-mediated isothermal amplification reactions with general PCR and real-time PCR. 5A to 5E are results of detecting intestinal bacteria producing carbapenem according to each sample sample. NDM-1 refers to the primer set in Table 7, and NDM-2 refers to the primer set in Table 8. The curve marked with ○ indicates a positive control, and the curve marked with X indicates a negative control.

Figure 6 is a result confirming the specificity of the detection of intestinal bacteria producing carbapenemase of the primer set for isothermal amplification reaction of the present invention. Curves marked with o indicate positive controls, curves marked with x indicate negative controls.

As described above, there is an urgent need to develop a method capable of examining the specificity and sensitivity of carbapenem-resistant intestinal bacteria at an early, rapid, accurate and quantitative basis. A method of detecting intestinal bacteria producing carbapenemase within 2 hours using Xpert (Cepheid) using real-time PCR is used, but in the case of Expert, expensive equipment and reagents are required.

Accordingly, in the present invention, a primer set for a loop-mediated isothermal amplification reaction for quickly and effectively detecting intestinal bacteria producing carbapenemase was prepared. A primer set for loop-mediated isothermal amplification reactions for IMP, VIM, KPC, and NDM, known as carbapenemase-generating genes, was prepared. The primer set showed sensitivity similar to that of a detection method using general PCR, and only 1 hour with simple equipment. It has the effect of efficiently detecting only intestinal bacteria producing carbapenemase within.

Therefore, the present invention (1) SEQ ID NO: 1 to SEQ ID NO: primer set for detecting IMP gene consisting of the base sequence of 6;

(2) a primer set for VIM gene detection consisting of the nucleotide sequence of SEQ ID NO: 15 to SEQ ID NO: 20;

(3) KPC gene detection primer set consisting of the nucleotide sequence of SEQ ID NO: 29 to SEQ ID NO: 34; And

(4) NDM gene detection primer set consisting of the nucleotide sequence of SEQ ID NO: 43 to SEQ ID NO: 48; Provided is a primer set for loop-mediated isothermal amplification (LAMP) for detecting carbapenemase producing enterobacteriaceae (CPE), which is composed of any one or more primer sets.

The "primer" of the present invention is a nucleic acid sequence having a short free 3'-hydroxyl group, which can form a complementary template and a base pair, and is a starting point for copying a template. A short nucleic acid sequence that functions. Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures and four different nucleoside triphosphates. PCR conditions, sense and antisense primer lengths can be modified based on those known in the art. These oligonucleotide primers can be easily prepared and used by those skilled in the art according to methods known in the art.

In addition, the "primer" of the present invention is a single-stranded oligonucleotide, suitable conditions (the presence of four different nucleoside triphosphates and polymerases such as DNA or RNA polymerase), suitable temperature and suitable Means acting as a starting point to initiate template-directed DNA synthesis under buffer. Oligonucleotides used as primers can also include nucleotide analogs, such as phosphorothioates, alkylphosphorothioates or peptide nucleicacids or intercalating agents. It may include.

The suitable length of the primer is determined by the properties of the primer to be used, but it can be used in a length of 8 to 500 bp. The primer need not be exactly complementary to the sequence of the template, but a complementary enough to form a hybridcomplex with the template can be used.

"Loop-mediated isothermal amplification (LAMP)" of the present invention is a method using a Bst DNA polymerase having a strand displacement DNA synthesis function, and two pairs of specific It is a method of continuously amplifying after forming a loop using a primer. As shown in the schematic diagram of Figure 1, forward primer (forward primer), reverse primer (backward primer), forward inner primer (Forward inner pimer; FIP), reverse inner primer (Back inner pimer; BIP), forward loop primer (LoopF) ) And a reverse loop primer (LoopB) to perform an amplification reaction using a primer set.

In the present invention, the primer set for IMP gene detection is a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, a forward inner primer of SEQ ID NO: 3 (FIP), a reverse internal primer of SEQ ID NO: 4 Back inner pimer (BIP), consisting of a forward loop primer (LoopF) of SEQ ID NO: 5 and a reverse loop primer (LoopB) of SEQ ID NO: 6,

The primer set for VIM gene detection includes a forward primer of SEQ ID NO: 15, a reverse primer of SEQ ID NO: 16, a forward inner primer of SEQ ID NO: 17 (FIP), and a reverse inner primer of SEQ ID NO: 18 (Back inner pimer; BIP) ), A forward loop primer of SEQ ID NO: 19 (LoopF) and a reverse loop primer of SEQ ID NO: 20 (LoopB),

The KPC gene detection primer set includes a forward primer of SEQ ID NO: 29, a reverse primer of SEQ ID NO: 30, a forward inner primer of SEQ ID NO: 31 (FIP), and a reverse inner primer of SEQ ID NO: 32 (Back inner pimer; BIP) ), A forward loop primer of SEQ ID NO: 33 (LoopF) and a reverse loop primer of SEQ ID NO: 34 (LoopB),

The NDM gene detection primer set includes a forward primer of SEQ ID NO: 43, a reverse primer of SEQ ID NO: 44, a forward inner primer of SEQ ID NO: 45 (FIP), and a reverse inner primer of SEQ ID NO: 46 (Back inner pimer; BIP). ), A forward loop primer of SEQ ID NO: 47 (LoopF) and a reverse loop primer of SEQ ID NO: 48 (LoopB).

In the present invention, the carbapenemase-producing enterococcal bacteria are Escherichia coli , Klebsiella pneumonia , Pseudomonas spp. , Serratia spp ., Citrobacter spp . ), Acinetobacter spp. It may be one or more selected from the group consisting of Morganella spp. , Providencia spp. , And Enterobacter spp. , Preferably, Escherichia coli , pneumococcal ( Klebsiella pneumonia ), Pseudomonas aeruginosa , Citrobacter freundii , Acinetobacter baumannii , Morganella morganii , Serratia marcescens , Serratia marcescens Expression of one or more genes of Enterobacter cloacae , Enterobacter aerogenes and Providencia stuartii , or IMP, VIM, KPC and NDM genes that produce carbapenemase Intestinal bacteria known to be capable of being detected without limitation.

In a specific embodiment of the present invention, in the present invention, two sets of primers for loop-mediated isothermal amplification reactions for IMP, VIM, KPC, and NDM are prepared, and sensitivity and efficiency for detecting intestinal bacteria produced by carbapenemase are produced. To select high primer sets, primer sets corresponding to Tables 1 to 8 were prepared. In addition, by comparing the primer set for loop-mediated isothermal amplification reaction and general PCR and real-time PCR, the primer set for Table 9 and Table 10 was prepared to compare the sensitivity and efficiency of detection of intestinal bacteria, thereby generating carbapenemase. Comparative experiments were performed using DNA isolated from intestinal bacteria.

As a result, as shown in Figures 2 to 5, as a result of confirming the sensitivity to detect carbapenemase-producing intestinal bacteria between two primer sets for isothermal amplification, primer sets corresponding to Table 1, Table 3, Table 5 and Table 7 It was confirmed that the sensitivity of detecting carbapenemase-producing intestinal bacteria is higher than the primer sets corresponding to Tables 2, 4, 6, and 8. That is, it was confirmed that the detection sensitivity was significantly different according to the primer sequence for the loop-mediated isothermal amplification reaction. In addition, as shown in Figures 2 to 5 and Table 13, it was confirmed that the primer set for isothermal amplification reactions exhibits similar sensitivity to general PCR.

Based on the above results, in the present invention, IMP loop-mediated isothermal amplification reaction primer set-1, VIM loop-mediated isothermal amplification reaction primer set-1, KPC loop-mediated isothermal amplification reaction primer set-1 and NDM loop-mediated isothermal amplification reaction It was confirmed that the primer set-1 was suitable for detecting intestinal bacteria producing carbapenemase.

In another specific embodiment of the present invention, to determine whether the loop-mediated isothermal amplification reaction primer set can actually specifically detect only carbapenemase-producing intestinal bacteria, various types of general strains, carbapenem-resistant intestinal bacteria ( Carbapenem resistant enterobacteriaceae (CRE), ESBL (Extended-spectrum beta-lactamases) and non-ESBL (non Extended-spectrum beta-lactamases) using isothermal amplification reaction in the same manner as in Example 2-2, As shown in FIG. 6, the reaction was observed only in the positive control group, and it was confirmed that there was no reaction in the non-carbapenem-producing intestinal bacteria. That is, it was confirmed that the primer set for the loop-mediated isothermal amplification reaction of the present invention can be clearly distinguished and detected by ESBL-producing intestinal bacteria and carbapenem-resistant intestinal bacteria and carbapenemase-producing intestinal bacteria.

The present invention also provides a kit for detecting carbapenemase-producing intestinal bacteria comprising a primer set for loop-mediated isothermal amplification (LAMP).

In the kit of the present invention, the reagent for performing the amplification reaction is not particularly limited as long as it is usually included in the kit, but preferably, a DNA polymerase for loop-mediated isothermal amplification reaction, dNTP (dATP, dCTP, dGTP and dTTP) Mixtures, buffer solutions, and distilled water.

In addition, the kit of the present invention may further include a user guide describing optimal conditions for performing the reaction. The handbook is a printout that describes how to use the kit, for example, how to prepare a buffer, and the reaction conditions presented. The guide may include a brochure or leaflet, a label attached to the kit, and a description on the surface of the package containing the kit. In addition, the handbook may include information disclosed or provided through an electric medium such as the Internet.

The present invention also

(A) separating the DNA from the sample;

(b) using the extracted DNA as a template,

(1) Primer set for IMP gene detection consisting of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 6;

(2) a primer set for VIM gene detection consisting of the nucleotide sequence of SEQ ID NO: 15 to SEQ ID NO: 20;

(3) KPC gene detection primer set consisting of the nucleotide sequence of SEQ ID NO: 29 to SEQ ID NO: 34; And

(4) NDM gene detection primer set consisting of the nucleotide sequence of SEQ ID NO: 43 to SEQ ID NO: 48; Amplifying a target sequence by performing a loop-mediated isothermal reaction using a primer set for any one or more of the loop-mediated isothermal reactions; And

(c) detecting the amplification product, it provides a method for detecting intestinal bacteria producing carbapenemase.

The step (a) is a step of separating DNA from a sample, wherein the sample is obtained by a fecal or rectal swab collected from an organism, and the sample is homogenized and extracted from the biological sample as necessary. It may be a sample obtained by performing any necessary pretreatment. Such pretreatment can be selected by a person skilled in the art depending on the biological sample to be targeted.

DNA obtained from the sample can be obtained using methods well known in the art, such as using a commercially available DNA extraction kit from a sample obtained from an individual using feces or rectal swab. have. When the amount of the nucleotide is small, an operation of amplifying the nucleotide can be performed as necessary. The amplification operation can be performed, for example, through a PCR method using an enzyme such as DNA polymerase.

Next, the step (b) is a step of performing a loop-mediated isothermal amplification reaction using DNA extracted from a sample as a template, using a primer set for loop-mediated isothermal amplification for detecting intestinal bacteria producing carbapenemase of the present invention. It is characterized in that it is carried out, preferably at 60 ℃ to 70 ℃, more preferably at 65 ℃.

The loop-mediated isothermal amplification reaction is similar to the conventional polymerase chain reaction (PCR) method, but the existing polymerase chain reaction repeatedly performs three steps of denaturation, conjugation, and elongation, thereby amplifying the target gene. While it is necessary to continuously change the temperature during the reaction process, the loop-mediated isothermal amplification reaction can be conjugated and stretched at a constant temperature. Since the loop-mediated isothermal amplification reaction uses Bst DNA polymerase having nucleic acid terminal hydrolase activity instead of Taq DNA polymerase, it is possible to induce denaturation of DNA's double helix structure without being dependent on heat.

Finally, step (c) is a step of detecting amplification products, and each of the IMP, VIM, KPC and NDM genes is specifically amplified by the loop-mediated isothermal amplification primer, and IMP, VIM, KPC and When any of the NDM genes is amplified, it can be determined that it is an intestinal bacterium producing carbapenemase.

Whether or not amplification products are generated can be detected by labeling a detectable labeling material on the amplification target sequence, and as one specific example, the labeling material may be a material emitting fluorescence, phosphorescence, or radiation, but is not limited thereto.

The labeling material can be identified by the naked eye, a sensor or other means, preferably the labeling material is biotin, digoxigenin, dinitrophenol, aminoacetylfluorene , Sulfonate, FITC (Fluorescein isothiocyanate), rhodamine, aminomethylcoumarin, FAM (Carboxyl fluorescein-aminohexyl-amidite), TAMRA (Tetramethyl-6-Carboxyrhodamine) and cyanine It may be selected from one or more, but if it can function as a cover, the type is not particularly limited.

Furthermore, the present invention provides a method for providing information on whether or not to administer carbapenem using the method for detecting intestinal bacteria producing carbapenemase.

When using the primer set for the loop-mediated isothermal amplification reaction of the present invention, if any of the IMP, VIM, KPC, and NDM genes is amplified, it can be determined that it is an intestinal bacterium producing carbapenemase, and in this case, carbapenem administration You can provide information to decide not to.

Hereinafter, the present invention will be described in more detail through examples.

These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1: Preparation of a primer set for loop-mediated isothermal amplification reaction for detection of intestinal bacteria producing carbapenemase

1-1: Loop-mediated isothermal amplification reaction primer set

In the present invention, a primer set for loop-mediated isothermal amplification reactions for IMP, VIM, KPC, and NDM known as carbapenemase generating genes was prepared.

The loop-mediated isothermal amplification (LAMP) method is a method using a Bst DNA polymerase having a strand displacement DNA synthesis function, and loops using two pairs of specific primers. It is a method of continuously amplifying after forming. In the present invention, in order to effectively perform loop-mediated isothermal amplification, as shown in the schematic diagram of FIG. 1, forward primer, backward primer, forward inner pimer (FIP), reverse inner primer ( A primer set consisting of a back inner pimer (BIP), a forward loop primer (LoopF) and a reverse loop primer (LoopB) was prepared.

In addition, a primer set for loop-mediated isothermal amplification reactions for IMP, VIM, KPC, and NDM was prepared in each of two sets to select a primer set with high sensitivity and efficiency for detection of intestinal bacteria producing carbapenemase. Primers were prepared by requesting Macrogen, and each primer sequence is shown in Tables 1 to 8 below.

1-2: Producing primers for general PCR and real-time PCR

In the present invention, compared to the primer set for the loop-mediated isothermal amplification reaction prepared in Example 1-1 and general PCR and real-time PCR, to compare the sensitivity and efficiency of detection of carbapenemase-producing intestinal bacteria, general PCR (Conventional PCR) and Primer sets for real-time PCR (Real-time PCR) were also prepared. For real-time PCR primer set for IMP gene detection, primers having the same nucleotide sequence as F3 and B3 in Table 1 were used.

Example 2: Detection of intestinal bacteria producing carbapenemase

2-1: DNA extraction

In the present invention, by using the loop-mediated isothermal amplification reaction primer set prepared in Example 1, a general PCR primer set, and a real-time PCR primer set, it was intended to detect intestinal bacteria producing carbapenemase. Samples were obtained using a rectal swab, and inoculated with the carbapenemase-producing enteric bacteria isolated from the samples were inoculated into MacConkey agar and cultured at 37 ° C. for over night. The cultured strain was adjusted to 1.5 x 10 ⁸ cells (MaF 0.5) using a turbidimeter, transferred to a 1.5 ml tube, boiled at 100 ° C for 10 minutes, and centrifuged at 13,000 rpm for 5 minutes. After centrifugation, supernatant containing DNA was obtained. The obtained DNA for 1.5 x 10 ⁸ cells was prepared by serial dilution to 1.5 x 10. As shown in Table 11, intestinal bacteria expressing IMP, VIM, KPC and NDM genes in the sample were isolated, and a positive control for each of the carbapenemase-generating genes was used.

2-2: Sensitivity and efficiency comparison of detection of intestinal bacteria producing carbapenemase according to detection method and primer type

In the present invention, using the DNA isolated in Example 2-1 as a template, a carbapenemase intestine is produced using the primer set for the loop-mediated isothermal amplification reaction prepared in Example 1, a general PCR primer set, and a real-time PCR primer set. Bacterial detection sensitivity was confirmed.

First, for general PCR, prepare a bionic premix PCR tube (Bioneer premix PCR tube; BIONEER AccuPower PCR PreMix, K-2016-C), and use the primer set for each gene in Table 9 according to the product manual. Amplified. For DNA amplification, 1 μl of Reactive Buffer (with 1.5 mM MgCl2) and 250 μM of dNTP each and 1 U of DNA polymerase ( Top DNApolymerase) are freeze-dried. A PCR reaction solution of 20 μl consisting of 16 μl and 2 μl of Example 2-1 DNA was prepared, after 5 minutes and 1 cycle reaction at 94 ° C. for 30 seconds at 94 ° C., 56 ° C. for the IMP gene, and 42 ° C. for the VIM gene. , KPC, NDM genes were repeated at 52 ° C for 45 seconds and 72 ° C for 1 minute treatment at 1 cycle. PCR was performed at 35 cycles for the IMP and VIM genes and 30 cycles for the KPC and NDM genes, and then each amplified product was amplified by electrophoresis (120 V / 35 min) using 1.5% agarose gel. .

Real-time PCR using Bio-rad's kit (Bio-rad SsoAdvanced Universal SYBR Green supermix kit; 172-5271) according to the product manual, real-time PCR analysis equipment (Bio-rad CFX connect Real-time system; 788BR06588 ), And the primer set for each gene in Table 10 was used. For DNA amplification, 10 µl of 2X superpremix (polymerase, dNTP, MgCl2, and SYBR Green 1 dye is included), 10 µl primer each, 1 µl, 6 µl of sterile tertiary distilled water, and 2 µl of Example 2-1 DNA A 20 μl PCR reaction solution was prepared, and after 3 minutes of reaction at 98 ° C., 40 cycles were performed with treatment at 98 ° C. for 10 seconds and 60 ° C. for 30 seconds for 1 cycle.

The loop-mediated isothermal amplification reaction was performed according to the product manual using the NEB LAMP kit (WarmStart LAMP kit; E1700L), and analysis was performed using a real-time PCR analysis device (Bio-rad CFX connect Real-time system; 788BR06588). DNA was amplified using a primer set for each gene in Tables 1 to 8, and a reaction solution was prepared as shown in Table 12, and amplification reaction was performed at 65 ° C for 60 minutes.

As a result, as shown in FIGS. 2 to 5, it was confirmed that the primer set for the loop-mediated isothermal amplification reaction showed sensitivity similar to that of a normal PCR. In addition, as a result of confirming the sensitivity of detecting carbapenemase-producing intestinal bacteria between two primer sets for loop-mediated isothermal reaction, primer sets corresponding to Table 1, Table 3, Table 5, and Table 7 are shown in Tables 2, 4, and 6 And it was confirmed that the sensitivity of detecting intestinal bacteria producing carbapenemase is higher than the primer set corresponding to Table 8. That is, it was confirmed that the detection sensitivity shows a large difference according to the primer sequence, and in the present invention, the primer sets corresponding to Table 1, Table 3, Table 5, and Table 7 having high detection sensitivity and general PCR and real-time PCR analysis sensitivity are shown below. It was summarized as Table 13.

Based on the above results, in the present invention, IMP loop-mediated isothermal amplification reaction primer set-1, VIM loop-mediated isothermal amplification reaction primer set-1, KPC loop-mediated isothermal amplification reaction primer set-1 and NDM loop-mediated isothermal amplification reaction It was confirmed that the primer set-1 was suitable for detecting intestinal bacteria producing carbapenemase.

Example 3: Confirmation of specificity of the primer set for loop-mediated isothermal amplification reaction

In the present invention, various types of general strains, carbapenem resistant intestinal bacteria (carbapenem resistant) in order to confirm that the primer set for loop-mediated isothermal amplification reaction which confirmed the effect in Example 2 can actually detect only carbapenemase-producing intestinal bacteria A loop-mediated isothermal amplification reaction was performed in the same manner as in Example 2-2 using enterobacteriaceae (CRE), ESBL (Extended-spectrum beta-lactamases) and non-ESBL (non Extended-spectrum beta-lactamases). The strain was obtained in the same manner as in Example 2-1, and each antibiotic resistance characteristic was identified and classified into carbapenem-resistant enterococcal bacteria, ESBL, and non-ESBL. As a positive control, the positive control shown in Table 11 above was used, respectively.

As shown in FIG. 6, the reaction was observed only in the positive control group, and it was confirmed that there was no reaction in the carbapenem-resistant strain and other betalactam-based antibiotic-resistant strains other than the carbapenemase-producing intestinal bacteria.

That is, it was confirmed that the loop-mediated isothermal amplification primer set of the present invention can selectively detect only intestinal bacteria producing carbapenemase.

The present invention confirmed that the primer set for loop-mediated isothermal amplification reaction for the detection of carbapenemase-producing intestinal bacteria can be carried out within 1 hour with simple equipment, effectively detecting carbapenemase-producing intestinal bacteria. Since it was confirmed, it can be usefully used as a diagnostic kit for detecting intestinal bacteria producing carbapenemase.

Claims (10)

1. (1) Primer set for IMP gene detection consisting of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 6;

   (2) a primer set for VIM gene detection consisting of the nucleotide sequence of SEQ ID NO: 15 to SEQ ID NO: 20;

   (3) KPC gene detection primer set consisting of the nucleotide sequence of SEQ ID NO: 29 to SEQ ID NO: 34; And

   (4) NDM gene detection primer set consisting of the nucleotide sequence of SEQ ID NO: 43 to SEQ ID NO: 48; A primer set for loop-mediated isothermal amplification (LAMP) for the detection of carbapenemase producing enterobacteriaceae (CPE), consisting of any one or more primer sets.
2. According to claim 1, The primer set for IMP gene detection is a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, a forward inner primer of SEQ ID NO: 3 (FIP), a reverse internal primer of SEQ ID NO: 4 (Back inner pimer; BIP), characterized in that consisting of a forward loop primer (LoopF) of SEQ ID NO: 5 and a reverse loop primer (LoopB) of SEQ ID NO: 6, loop-mediated isothermal amplification for detection of intestinal bacteria producing carbapenemase Primer set for reaction.
3. The primer set for detecting a VIM gene is a forward primer of SEQ ID NO: 15, a reverse primer of SEQ ID NO: 16, a forward inner primer of SEQ ID NO: 17 (FIP), and a reverse internal primer of SEQ ID NO: 18. (Back inner pimer; BIP), characterized in that consisting of a forward loop primer (LoopF) of SEQ ID NO: 19 and a reverse loop primer (LoopB) of SEQ ID NO: 20, loop-mediated isothermal amplification for detection of intestinal bacteria producing carbapenemase Primer set for reaction.
4. The primer set for detecting a KPC gene is a forward primer of SEQ ID NO: 29, a reverse primer of SEQ ID NO: 30, a forward inner primer of SEQ ID NO: 31 (FIP), a reverse internal primer of SEQ ID NO: 32. (Back inner pimer; BIP), characterized in that consisting of a forward loop primer (LoopF) of SEQ ID NO: 33 and a reverse loop primer (LoopB) of SEQ ID NO: 34, loop-mediated isothermal amplification for the detection of intestinal bacteria producing carbapenemase Primer set for reaction.
5. The primer set for NDM gene detection is a forward primer of SEQ ID NO: 43, a reverse primer of SEQ ID NO: 44, a forward inner primer of SEQ ID NO: 45 (FIP), a reverse internal primer of SEQ ID NO: 46. (Back inner pimer; BIP), characterized in that consisting of a forward loop primer (LoopF) of SEQ ID NO: 47 and a reverse loop primer (LoopB) of SEQ ID NO: 48, loop-mediated isothermal amplification for detection of intestinal bacteria producing carbapenemase Primer set for reaction.
6. The method of claim 1, wherein the intestinal bacterium producing carbapenemase is Escherichia coli , Klebsiella pneumonia , Pseudomonas spp. , Serratia spp ., Citrobacter spp. ), Acinetobacter spp. A loop for detecting carbapenemase-producing enterococcus, characterized by at least one selected from the group consisting of Morganella spp. , Providencia spp. , And Enterobacter spp. Primer set for each isothermal amplification reaction.
7. A kit for detecting carbapenemase-producing intestinal bacteria, comprising the primer set for loop-mediated isothermal amplification reaction for detecting carbapenemase-producing intestinal bacteria of claim 1.
8. According to claim 7, The kit is characterized in that it further comprises a loop-mediated isothermal amplification reaction DNA polymerase, dNTP (dATP, dCTP, dGTP and dTTP) mixture, buffer solution and distilled water, carbapenemase Kit for detecting intestinal bacteria.
9. A method for detecting intestinal bacteria producing carbapenemase comprising the step of amplifying the carbapenemase generating gene using the primer set for isothermal amplification according to claim 1.
10. The method of claim 9, wherein the amplification reaction is performed at 60 ° C to 70 ° C, a method for detecting intestinal bacteria producing carbapenemase.

Images