KR20090114273A - Kit for detecting Mycobacterium leprae and detection method thereof - Google Patents

Abstract

PURPOSE: A kit and a method for diagnosing leprosy are provided to detect Mycobacterium leprae with improved sensitivity and specificity. CONSTITUTION: A kit for diagnosing leprosy comprises a tube containing R1(5'-CGGGGTAGGGGCGTTTTAGT-3'; sequence number 1), R2(5'-CTAGAAGGTTGCCGTATGTG-3'; sequence number 2), R3(5'-GCGTTTTAGTGTGCATGTCA-3'; sequence number 3), and R4(5'-GGATCATCGATGCACTGTTC-3'; sequence number 4) primers. A method for diagnosing leprosy comprises: a step of preparing DNA sample from an animal; a step of performing PCR using R1(5'-CGGGGTAGGGGCGTTTTAGT-3';sequence number 1), R2(5'-CTAGAAGGTTGCCGTATGTG-3';sequence number 2), R3(5'-GCGTTTTAGTGTGCATGTCA-3';sequence number 3) and R4(5'-GGATCATCGATGCACTGTTC-3';sequence number 4); and a step of isolating amplified PCR product.

Description

Kit for detecting Mycobacterium leprae and detection method

The present invention relates to a kit for diagnosing leprosy and a diagnostic method thereof. More specifically, the present invention relates to a leprosy diagnostic kit capable of detecting mycobacterium refre, which is a causative agent of leprosy.

Mycobacterium leprae (hereinafter referred to as M.leprae) is an obligatory intracellular parasite and is a etiological factor of leprosy. Although the incidence of leprosy patients decreases every year, the need for early diagnosis of leprosy is urgently needed because once it occurs, treatment takes considerable time and money.

Detection of leprosy is important for the diagnosis of leprosy, but it is not based on the presence or absence of microorganisms on the microscope by acid-fast bacilli (AFB) in the skin smear. The extent of treatment is determined by calculation of the bacterial index (BI) and morphological index (MI). The diagnosis of Hansen's disease is also the basic method of diagnosis based on the clinical findings of AFB positive and skin lesions and nerve damage (Santo, AR, et al., J. Med.Microbiol., 39: 298-304, 1993). However, when the bacteria index of 0 is often difficult to make a diagnosis, wherein 10 of the even uniform showing positive in acidic dye can be confirmed by Mycobacterium leprae there is no minimum number of bacteria that can be observed with the bacteria on microscopic tissue ⁴ / A significant amount of g should be present, and it is difficult to distinguish the species if it is mixed with other antibacterial bacteria belonging to non-tuberculous mycobacteria (NTM) (Donoghue HD, et al, J. Med. Microbiol., 50: 177-182, 2001; Kang, TJ, et al., Clin.Exp.Dermatol., 28: 420-424, 2003; Kurabachew M, et al., J. Clin.Microbiol., 36: 1352-1356, 1998 Yoon, KH, et al., Kor. J. Dermatol., 32: 409-415, 1994). Therefore, the diagnosis of leprosy suffers a great deal if there are no bacteria found in the histological examination, the inflammatory response is non-specific, and there is no neurological inflammation.

In the diagnosis of leprosy, we tried to detect and isolate leprosy in patients using serological and molecular markers. It is difficult to detect bacillus because it can be detected and identified according to any potential body's immune defense state, and histological findings are also nonspecific. Since this method is low in specificity and sensitivity, in order to detect the bacterium in the presence of a low cell count, a variety of genomic sequences, namely 65 kDa antigen gene, 18 kDa antigen gene, 36 kDa antigen gene, Amplification of DNA through polymerase chain reaction using repetitive sequences is widely used.

In order to solve this problem, recently, a polymerase chain reaction (PCR) is used to amplify specific DNA sequences of leprosy, which is one of molecular biological diagnostic methods, to confirm the presence or absence of leprosy in tissues. In the double polymerase chain reaction, amplify the gene to be amplified by outside primers first, and then react with inside primers targeting a part of the nucleotide sequence of the reaction product obtained therein, and then perform a second amplification to use various kinds of samples of the patient. This is a diagnostic method that can directly isolate nucleic acid of Na bacteria and selectively amplify only nucleic acid of the desired site from among them, and can detect the small number of bacteria quickly and accurately. The conventional polymerization using only one pair of primers in terms of specificity and sensitivity Superior to enzymatic chain reactions (see Chae, GT, et al., J. Med. Microbiol., 51: 417-422, 2002; Jeon, JH, et al., Kor. Leprosy Bulletin, 30: 81-87, 1997; Kampirapap, K, et al, Int. J. Lepr.Other Mycobact Dis., 66: 16-21, 1998; Kang, TJ, et al., Clin.Exp.Dermatol., 28: 420 -424, 2003; Lee, TK, et al., J. Pusan Medical college., 33: 265-274, 1993; Patro cinio, LG, et al., FEMS Immunol.Med.Microbiol., 44: 311-316, 2005; Plikaytis, BB, et al., J. Clin Microbiol., 28: 1913-1917, 1990; Santos, AR, et al., J. Med. Microbiol., 39: 298-304, 1993; Scollard, DM, et al., Am J. Clin.Pathol., 109: 642-646, 1998; Torres, P., et al , Lepr. Rev., 74: 18-30, 2003; Yoon, KH, et al., Kor. J. Dermatol., 32: 409-415, 1994).

Accordingly, the present inventors have made intensive studies to develop a kit capable of more effectively diagnosing leprosy, and as a result, when using a double polymerase chain reaction kit including two pairs of primers, a conventional pair of primers is included. The specificity and sensitivity of the polymerase chain reaction kit was improved, and it was confirmed that leprosy could be efficiently diagnosed, and thus the present invention was completed.

In particular, the present invention is to provide a new method for diagnosing leprosy by observing the detection rate of leprosy using one-tube nested PCR to amplify by putting two pairs of primers in the same tube and comparing the results of PCR with other diagnostic methods.

In order to achieve the above object, the present invention provides R1 (5'-CGGGGTAGGGGCGTTTTAGT-3 '; SEQ ID NO: 1), R2 (5'-CTAGAAGGTTGCCGTATGTG-3'; SEQ ID NO: 2), R3 (5'-GCGTTTTAGTGTGCATGTCA-3 '; Provided is a kit for diagnosing leprosy bacteria comprising a tube comprising SEQ ID NO: 3) and R4 (5'-GGATCATCGATGCACTGTTC-3 '; SEQ ID NO: 4) primer.

In a preferred embodiment of the invention, it is preferred that there is one tube.

In the present invention, the leprosy bacteria is preferably Mycobacterium leprae, but is not limited thereto.

The present invention also comprises the steps of (a) obtaining a genomic DNA sample from an animal; (b) R1 (5'-CGGGGTAGGGGCGTTTTAGT-3 '; SEQ ID NO: 1), R2 (5'-CTAGAAGGTTGCCGTATGTG-3'; SEQ ID NO: 2), R3 (5'-GCGTTTTAGTGTGCATGTCA-3 '; SEQ ID NO: 3 for the sample; ) And performing a polymerase chain reaction (PCR) using R4 (5′-GGATCATCGATGCACTGTTC-3 ′; SEQ ID NO: 4) primers; And (c) separating the amplified PCR product.

In the preferred diagnostic method of the present invention, the primer is preferably carried out a polymerase chain reaction in one tube.

In addition, it is preferable that the animal of the present invention is a human (if there are other animals that can be leprosy in addition to human, please describe) leukemia diagnostic method characterized in that.

In the method of the present invention, the leprosy bacteria is preferably Mycobacterium leprae, but is not limited thereto.

In the present invention, cells were obtained from a sample, nucleic acids were extracted from the cells, and PCR amplification was performed. Methods of extracting nucleic acids from cells and PCR methods were performed through methods well known in the art, such as those described in Molecular cloning (1989) by Sambrook et al.

The nucleic acid amplification method of the present invention used a primer. Generally, the primer is preferably about 10 to 25 bp in length oligonucleotide, but may be longer. The primer is preferably single stranded but may be present in double or single stranded form. It described in the Example.

Primers of the invention have been prepared by conventional oligonucleotide synthesis methods, such synthesis methods are disclosed in US Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, and 5,602,244.

Sequences such as primers of the invention can be used to form a double helix optionally with complementary sequences, such as DNA derived from a sample. One skilled in the art can alter the hybridization conditions to change the selectivity of the primer for the target sequence.

In some embodiments, nucleic acids of defined sequences of the invention may be employed in combination with appropriate means such as labels to determine hybridization. Suitable labeling means are various labeling means including fluorescence, radioisotope enzymes or other ligands.

Various template dependent processes are possible to amplify specific gene products present in a given cell sample. One of the most known amplification methods is polymerase chain reaction (PCR), which is described in US Pat. Nos. 4,683,202 and 4,800,159 and the like.

It is generally desirable to separate the amplification product from the template or excess primer or other amplification product in one or another step. For example, the amplification products can be separated using agarose, agarose-acrylamide or PAGE using the standard method described in Molecular cloning (1989) by Sambrook et al.

Chromatography, such as adsorption, partitioning and ion exchange, can also be employed as an effective separation method. These separation methods can be adapted to function in clinical settings to process large volumes of samples, and new methods for separating or detecting thousands of samples at once, FMAT (fluorometric microvolume assay technique), chemiluminescence, and sequence detection systems (Applied) Biosystems) and mass spectrometry.

Nucleic acids according to the invention will be detected and quantified. In one embodiment the detection can be performed by visual means. Typical visual means method is to dye the gel with ethidium bromide and visualize the bands under UV light. If the amplification product is nucleotides labeled with radiation or fluorescence, the isolated amplification product performs radiosynthesis or fluorescent detection of the inserted radioisotope label.

The present invention includes a kit for carrying out the method. In a non-limiting embodiment the kit of the invention may comprise primers, enzymes for amplification and additional reagents. Thus, the kits will include these one or more reagents in suitable container means. The kits may also include reagents for isolating nucleic acids and purifying amplification products. The components of the kits may be packaged in an aqueous medium or lyophilized form, and suitable container means of the kit include at least one vial, test tube, flask, bottle, etc. into which the components may be placed. If one or more components are present in the kit, they may include other additional containers, such as second, third, etc., where the additional components may be located separately. However, a combination of components may be included in one container.

Hereinafter, the present invention will be described.

In the present invention, the RLEP primer pair prepared for the specific diagnosis of leprosy only reacted specifically with M. leprae in both one-tube nested PCR, and 15 kinds of Mycobacterium including M. tuber-culosis used as a control group. Did not respond. From these results, it was confirmed that the PCR method using the RLEP primer pair has high diagnostic specificity.

In addition, in the comparative analysis of the sensitivity of the standard strain of M. leprae , it was also confirmed that the diagnostic method using one-tube nested PCR was about 100 times more sensitive than the diagnostic method using single PCR.

Two-tube nested PCR usually has a very limited amount of DNA in the sample, so if a single PCR cannot be detected, a second PCR is performed using the inner primer. Two primers must be attached, the first polymerase chain reaction using outer primers, and the second amplification with the product from which the new reagent is added, the reaction can occur smoothly. It has the advantage of improving the sensitivity and specificity compared to the conventional PCR by a small number of reactions, but the problem of contamination often occurs through two amplification process by changing the tube, one tube to solve this problem Try one-tube nested PCR by putting 2 pairs of primers together to reduce contamination In part, the results were better than the conventional two-tube PCR.

Therefore, in the present invention, the two-tube nested PCR using two separate PCR strains to perform one PCR, but the method of detection of bacteria using one-tube nested PCR, which is a method of sequentially amplifying two pairs of primers in the same tube The sensitivity and specificity of were compared with the conventional PCR and single- and two-tube methods to evaluate the usefulness of one-tube nested PCR in the diagnosis of leprosy.

As described and demonstrated in detail above, the present invention provides a leprosy diagnostic kit capable of detecting mycobacterium refre, which is a causative agent of leprosy. The leprosy diagnostic kit of the present invention can be used to diagnose leprosy with improved specificity and sensitivity as compared to a conventional polymerase chain reaction kit, and thus it may be widely used for early diagnosis of leprosy.

Therefore, one-tube nested PCR method using repetitive sequence of bacillus in case of detection of bacteria in skin biopsy tissue due to anti-acid staining, in case of small number of pathogens to be detected or when a large number of samples should be processed simultaneously Is fast, bacterium specific, and has high sensitivity, which is considered to be useful in diagnosing bacterium.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example  1: Lactobacillus and In biopsy tissue DNA Extraction of

M. leprae strain 4264, used as a standard strain, was supplied with 100 mg (including an average of 2.9 × 10 ⁹ bacilli / mg) purified from Colorado State University using QIAamp DNA Mini Kit (Tissue protocol; Qiagen GmbH, Hilden, Germany). DNA was isolated and the final concentration was used at 50 ng / μl. The DNA used for the evaluation of clinical specimens was obtained by using DNA extracted from the leprosy patients from the leprosy patients using the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany).

Example  2: From antibacterial bacteria DNA Extraction of

M. avium , M. kansasii , M. chelonae , M. intracellulara , M. terrae , M. triviale, M. scrofulaceum , M. celatum , M. szulgai , M. africarum , M. marinum , M. mucogenicum , M. abscessus , M. tuberculosis H37RV and M. gortuitum were used at the Yonsei University College of Clinical Pathology. Reaction was carried out for 24 hours. Cetyl Trimethyl Ammonium Bromide (CTAB) was added and reacted at 65 ° C. for 10 minutes, followed by phenol treatment and ethanol precipitation to obtain DNA.

Example 3: PCR

Among the repetitive sequences of the bacterium DNA (GenBank Accession No. AL583917), a similar base sequence was extracted through a Blast search provided by the National Center for Biotechnology Information (NCBI) and analyzed using the MultAlin program. As a result, R1 (5'-CGGGGTAGGGGCGTTTTAGT-3 '; SEQ ID NO: 1) and R2 (5'-CTAGAAGGTTGCCGTATGTG-3'; SEQ ID NO: 2) and the expected amplification products were 272-bp and 230-bp, respectively. Two pairs of primers R3 (5'-GCGTTTTAGTGTGCATGTCA-3 '; SEQ ID NO: 3) and R4 (5'-GGATCATCGATGCACTGTTC-3'; SEQ ID NO: 4) were prepared (Bioneer, Daejeon, Korea) (Figure 1).

As a two-tube nested PCR reaction solution, Bioneer premix, 10 pmol of primer and template DNA of 5 μl were added in 20 μl of total. As the reaction conditions, pre-denaturation was performed once using an outer primer (R1-R2) for 5 minutes at 94 ° C, followed by 30 seconds denaturation at 94 ° C, 30 seconds annealing at 51 ° C, and 30 seconds extension at 72 ° C. Repeated 10 times, post-extension was performed at 72 ° C for 10 minutes, pre-denaturation was performed once at 94 ° C for 5 minutes using inner primer (R3-R4), followed by denaturation at 94 ° C for 30 seconds, and 58 ° C. 30 seconds annealing, repeated 35 times with a 30-second extension at 72 ℃, post-extension 10 minutes at 72 ℃.

One-tube nested PCR uses primers (R1, R2, R3, R4) to perform pre-denaturation once at 94 ° C for 5 minutes, followed by 30 seconds denaturation at 94 ° C, 30 seconds annealing at 65 ° C, and 72 ° C. After 15 times with a 60 second extension, 35 times were repeated at 94 ° C 30 seconds, 52 ° C 30 seconds, and 72 ° C 60 seconds, and post-extension was performed at 72 ° C for 10 minutes.

The single PCR used for the comparative experiment was Touch-Down (TD) PCR, and the F (5'-TGCATGTCATGGCCTTGAGG-3 '; SEQ ID NO: 5) and R (5'-CACCGATACCAGCGGCAGAAA) were expected to be 129-bp in size. -3 '; SEQ ID NO: 6) was used. As the reaction conditions, first, pre-denaturation was performed once at 94 ° C. for 5 minutes, followed by 7 times of 45 seconds denaturation at 94 ° C., decreasing at 1 ° C. from 64 ° C. to 58 ° C., and then again at 94 ° C. at 45 ° C. Ultra-denaturation, annealing for 45 seconds at 58 ℃, and 90 times extension at 72 ℃ was performed 35 times.

Gene amplifiers were used GeneAmp 2700 (Applied Biosystems, USA). The amplified gene product was isolated by electrophoresis on 2% agarose gel and stained with DNA with ethidium bromide (EtBr, Sigma, USA). PCR amplification and the size of the amplification product were observed using Gel Doc EQ (Bio Rad, USA), and 100 bp DNA ladder (Bioneer, Daejeon, Korea) was used as DNA size marker.

The result of the above example is as follows.

One - tube nested PCR and Single PCR M. leprae  Detection sensitivity

M. leprae for comparative analysis of the sensitivity of one-tube nested PCR and single PCR Strain 4264 DNA was serially diluted 10-fold and used as template DNA for PCR. As a result, the single PCR was amplified in more than 1 pg, one tube nested PCR was confirmed that the RLEP gene is amplified in more than 1 fg (Fig. 2). Therefore, the diagnostic method using one tube nested PCR was about 100 times more sensitive than the diagnostic method using single PCR.

One - tube nested PCR M. leprae  Detection specificity

15 Mycobacterium to investigate whether the RLEP primer pair designed to detect leprosy specifically reacts with M. leprae One-tube nested PCR was performed on control DNA isolated from spp. As a result, the RLEP gene fragment was specifically amplified only in the bacterium, but not in the DNA used as a control (FIG. 3).

In clinical samples Single PCR and One - tube nested PCR Detection efficiency

Specific gene detection of RLEP was performed from DNA extracted from skin tissue of leprosy patients. In general, the amount of DNA amplified by the one-tube nested PCR method was larger than that detected by the single PCR method, which was more clearly observed than the DNA amplified by electrophoresis (FIG. 4).

In addition, the bacterial index (Bacterial index, BI) in the following table of the present invention as a result of the staining after acidic bacteria, 1-3 is 100 to 10, 10, or on average one to ten of the bacterium is observed in one view respectively. 4-6 is a value which is judged when 10 to 1000 or more bacteria are observed in the average field of view. The Bacterial Index is usually obtained by staining antibacterial bacteria with six tissues, including both earlobe and representative lesions, and the basic method of diagnosis is as follows. When observed in the mean field, when 5+ 100 to 1000 strains are observed in the mean field, 4+ 10 to 100 strains are observed in the mean field, and 3+ 1 to 10 strains are observed in the mean field. When 10+ bacteria in 2 + 1 are observed in 10 visual fields, when 10+ bacteria in 1 + 1 are observed in 100 visual fields.

A bacterial index of 0 means that the result was not observed in the visual field after staining with acidophilic bacteria, but in the case of tuberculosis type or patients, the bacterial index was negative from the beginning. If the bacterial index is positive, it can be said that Hansen's disease.

However, in spite of the negative microscopic bacterial index, tuberculosis-type B with the bacterium present in the body means that the bacterial bacterium exists due to the limit of the bacterial index, which can be diagnosed as Hansen's disease. Therefore, although the bacterial index decreases, the time taken to reach the bacterial index to zero in patients with bacterial index 5+ should be about 5-6 years.

According to histopathologic findings, a total of 76 samples divided into 19 cases of bacterial index 0 and 23 cases of 1-3 and 34 cases of 4-6 were compared with PCR results (see Table 1).

The following table compares PCR methods for detecting M. leprae from biopsy samples of leprosy patients.

                    PCR BI (No. of sample) Single PCR positive One-tube nested PCR positive Two-tube nested PCR positive 0 (19) 5 (23.8%) 16 (84.2%) 12 (63.2%) 1-3 (23) 4 (17.4%) 21 (91%) 18 (78.3%) 4-6 (34) 16 (47.1%) 33 (97.1%) 30 (88.2%) total (76) 25 (32.1%) 70 (92.1%) 61 (79%)

As a result, the bacterial index was positive in 5 (23.8%) for single PCR, 4 (17.4%) for 1-3, and 16 (47.1%) for 4-6 and 12 (two-tube respectively). 63.2%), 18 (78.3%), 30 (88.2%), and 61 (79%), whereas one-tube nested PCR showed 16 (84.2%) and 1-3 for bacteria In 21 (91%) and 4-6 cases, 33 (97.1%) showed positive results.

1 is a diagram showing the position of the primer set for one-tube nested PCR and single PCR;

2 shows the reference strain M. leprae (Strain 4264) Comparison of sensitivity of (A) one-tube nested PCR and (B) single PCR using DNA, M, 100 bp DNA ladder (Bioneer, Daejeon, Korea), lanes 1, 100 pg, lanes 2, 10 pg, lane 3, 1 pg, lane 4, 100 fg, lane 5, 10 fg, lane 6, 1 fg, lane 7, 100 ag, N, negative control;

Figure 3 M. leprae in one tube nested PCR Photo showing the specificity of the fryers targeting the RLEP sequence. M, 100 bp DNA ladder (Bioneer, Daejeon, Korea), lane 1, H37RV, lane 2, M. avium , lane 3, M. chelonae , lane 4, M. intracellulare , lane 5, M. kansasii , lane 6, M. scrofulaceum , lane 7, M. terrae , lane 8, M. triviale , lane 9, M. africanum , lane 10, M. celatum , lane 11, M. marinum , lane 12, M. szulgai , lane 13, M . mucogenicum, lane 14, M. abscessus, lane 15, M. fortuitum, lane 16, M. leprae, N, represents a negative control;

Figure 4 is a photograph of the detection of leprosy using (A) one-tube nested PCR and (B) single PCR from a sample isolated from leprosy patients (lane 1 ~ 21). N, negative control; P, positive control; M, 100 bp DNA ladder (Bioneer, Daejeon, Korea).

<110> Molecules and Diagnostics Inc. <120> Kit for detecting Mycobacterium leprae and detection method          about <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R1 Primer <400> 1 cggggtaggg gcgttttagt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R2 Primer <400> 2 ctagaaggtt gccgtatgtg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R3 primer <400> 3 gcgttttagt gtgcatgtca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R4 primer <400> 4 ggatcatcga tgcactgttc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer <400> 5 tgcatgtcat ggccttgagg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer <400> 6 caccgatacc agcggcagaa 20  

Claims (7)

R1 (5'-CGGGGTAGGGGCGTTTTAGT-3 '; SEQ ID NO: 1), R2 (5'-CTAGAAGGTTGCCGTATGTG-3'; SEQ ID NO: 2), R3 (5'-GCGTTTTAGTGTGCATGTCA-3 '; SEQ ID NO: 3) and R4 (5'- GGATCATCGATGCACTGTTC-3 '; SEQ ID NO: 4) A kit for diagnosing leprosy bacteria comprising a tube comprising a primer. The kit for diagnosing leprosy bacteria according to claim 1, wherein the tube is one. The kit for diagnosing leprosy bacterium according to claim 1 or 2, wherein the leprosy is Mycobacterium leprae. (a) obtaining a DNA sample from the animal; (b) R1 (5'-CGGGGTAGGGGCGTTTTAGT-3 '; SEQ ID NO: 1), R2 (5'-CTAGAAGGTTGCCGTATGTG-3'; SEQ ID NO: 2), R3 (5'-GCGTTTTAGTGTGCATGTCA-3 '; SEQ ID NO: 3 for the sample; ) And performing a polymerase chain reaction (PCR) using R4 (5′-GGATCATCGATGCACTGTTC-3 ′; SEQ ID NO: 4) primers; And (C) leprosy diagnostic method comprising the step of separating the amplified PCR product. The method of claim 4, wherein the primer is a polymerase chain reaction performed in one tube. The method of claim 4 or 5, wherein the animal is a human. The method for diagnosing leprosy bacterium according to claim 4 or 5, wherein the leprosy is Mycobacterium leprae.

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