Method for detecting non-tuberculous mycobacteria, primer set and kit thereof

Abstract

A method for detecting non-tuberculous mycobacteria, comprising the following steps: providing a sample; providing a primer pair, the primer pair being selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a sequence having about 70% to about 99% homology with SEQ ID NO:1, a sequence having about 70% to about 99% homology with SEQ ID NO:2, the complementary strand of SEQ ID NO:1, and the complementary strand of SEQ ID NO:2; performing a polymerase chain reaction on the sample with the primer pair to obtain a product; and analyzing the product to detect the presence of non-tuberculous mycobacteria. Another provided is a kit for detecting non-tuberculous mycobacteria, comprising the aforementioned primer pair.

Description

Technical Field

[0001] The present disclosure relates to a detection method, particularly a method for detecting non-tuberculous mycobacteria, a primer set, and a kit thereof.

Prior Art

[0002] Mycobacteria can be divided into the Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM). Non-tuberculous mycobacteria refer to mycobacteria other than Mycobacterium tuberculosis and Mycobacterium leprae.

[0003] Since the symptoms of pulmonary infection with non-tuberculous mycobacteria are similar to those of tuberculosis, it is often misdiagnosed as tuberculosis. Due to the cumbersome and time-consuming processes of traditional methods for bacterial species identification and drug susceptibility testing, it is easy to delay the treatment of patients. Therefore, the differential test between non-tuberculous mycobacteria and the Mycobacterium tuberculosis complex is of great significance for early diagnosis, early treatment, and control of the infection risk. In view of this, there is an urgent need for a detection method that can clinically detect whether a patient is infected with the pathogen of the Mycobacterium tuberculosis complex at an early stage, which is very important for improving the cure rate of the disease.

Summary of the Invention

[0004] One embodiment of the present disclosure provides a method for detecting non-tuberculous mycobacteria, comprising the following steps: providing a sample; providing a primer pair selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a sequence having about 70% to about 99% homology with SEQ ID NO:1, a sequence having about 70% to about 99% homology with SEQ ID NO:2, the complementary strand of SEQ ID NO:1, and the complementary strand of SEQ ID NO:2; performing a polymerase chain reaction with the primer pair and the sample to obtain a product; and analyzing the product to detect the presence of non-tuberculous mycobacteria.

[0005] In one embodiment, the step of providing a sample comprises providing a specimen containing non-tuberculous mycobacteria.

[0006] In one embodiment, the specimen is one or a combination of blood, sputum, bronchoalveolar lavage fluid, urine, and feces.

[0007] In one embodiment, the step of performing a polymerase chain reaction with a primer pair and a sample to obtain a product includes performing a polymerase chain reaction such that the primer pair amplifies a partial sequence of the 16S ribosomal ribonucleic acid (16S rRNA) gene sequence in non-tuberculous mycobacteria to obtain a product, wherein the partial sequence is SEQ ID NO:5.

[0008] In one embodiment, the method for detecting non-tuberculous mycobacteria further includes providing at least one probe selected from the group consisting of SEQ ID NO:3, a sequence having about 70% to about 99% homology with SEQ ID NO:3, the complementary strand of SEQ ID NO:3, SEQ ID NO:4, a sequence having about 70% to about 99% homology with SEQ ID NO:4, and the complementary strand of SEQ ID NO:4; performing a polymerase chain reaction with the primer pair, the probe and the sample to obtain a product.

[0009] In one embodiment, in the step of performing a polymerase chain reaction with the primer pair, the probe and the sample to obtain a product, the polymerase chain reaction is a real-time quantitative polymerase chain reaction.

[0010] Another embodiment of the present disclosure is to provide a kit for detecting non-tuberculous mycobacteria, including a primer pair selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a sequence having about 70% to about 99% homology with SEQ ID NO:1, a sequence having about 70% to about 99% homology with SEQ ID NO:2, the complementary strand of SEQ ID NO:1, and the complementary strand of SEQ ID NO:2.

[0011] In one embodiment, the primer pair is SEQ ID NO:1 and SEQ ID NO:2.

[0012] In one embodiment, the kit for detecting non-tuberculous mycobacteria further includes a specimen, wherein the specimen is one or a combination of blood, sputum, bronchoalveolar lavage fluid, urine, and feces.

[0013] In one embodiment, the kit for detecting non-tuberculous mycobacteria further includes a target gene, wherein the target gene is the 16S ribosomal RNA sequence of non-tuberculous mycobacteria.

[0014] In one embodiment, the kit for detecting non-tuberculous mycobacteria further includes a template having a length of about 100 base pairs to about 250 base pairs.

[0015] In one embodiment, the template is SEQ ID NO:5.

[0016] In one embodiment, the kit for detecting non-tuberculous mycobacteria further comprises at least one probe selected from the group consisting of SEQ ID NO:3, a sequence having about 70% to about 99% homology with SEQ ID NO:3, the complementary strand of SEQ ID NO:3, SEQ ID NO:4, a sequence having about 70% to about 99% homology with SEQ ID NO:4, and the complementary strand of SEQ ID NO:4.

[0017] Another embodiment of the present disclosure provides a primer set, comprising: a forward primer selected from the group consisting of SEQ ID NO:1, a sequence having about 70% to about 99% homology with SEQ ID NO:1, and the complementary strand of SEQ ID NO:1; and a reverse primer selected from the group consisting of SEQ ID NO:2, a sequence having about 70% to about 99% homology with SEQ ID NO:2, and the complementary strand of SEQ ID NO:2.

[0018]

Brief Description of the Drawings

[0019] Please read the following detailed description in conjunction with the accompanying drawings, and the aspects of the present disclosure will be more easily understood. However, it should be noted that in accordance with the standard practice in this industry, the various features are not drawn to scale. In fact, the dimensions of the various features may be arbitrarily enlarged or reduced for the sake of clear discussion.

[0020] Figure 1 According to some embodiments of the present disclosure, it shows partial fragments of the 16S rRNA gene sequence, the positions of primer pairs and probe design.

[0021] Figure 2 According to some embodiments of the present disclosure, it is an electrophoresis result diagram of detection with primer pairs SEQ ID NO:1 and SEQ ID NO:2.

[0022] Figure 3 According to some embodiments of the present disclosure, it is an amplification curve diagram of performing real-time quantitative polymerase chain reaction under the condition of different template amounts.

[0023] Figures 4 to 12 According to some embodiments of the present disclosure, it tests the R 2 value obtained by performing real-time quantitative polymerase chain reaction on 9 non-tuberculous mycobacteria under the condition of different template amounts.

[0024] Figure 13 According to some embodiments of the present disclosure, it is an amplification curve diagram of performing real-time quantitative polymerase chain reaction in the case of 82 clinical samples.

Embodiments

[0025] To make the description of the present disclosure more detailed and complete, the following provides an illustrative description of the embodiments and specific examples of the present invention, but this is not the only form for implementing or applying the specific examples of the present invention. Each of the embodiments disclosed below can be combined or substituted with each other under beneficial circumstances, or other embodiments can be added to one embodiment without further record or explanation. In the following description, many specific details will be described in detail to enable the reader to fully understand the following embodiments. However, the embodiments of the present invention can also be practiced without such specific details.

[0026] In this document, unless the article is specifically limited in the text, "a" and "the" can generally refer to a single one or more. It will be further understood that the terms "comprising", "including", "having" and similar terms used herein specify the features, regions, integers, steps, operations, components and / or groups thereof recorded, but do not exclude other features, regions, integers, steps, operations, components, components, and / or groups thereof.

[0027] An embodiment of the present disclosure provides a method for detecting non-tuberculous mycobacteria, comprising the following steps: providing a sample and providing a primer pair. The primer pair is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a sequence having about 70% to about 99% homology with SEQ ID NO:1, a sequence having about 70% to about 99% homology with SEQ ID NO:2, the complementary strand of SEQ ID NO:1, and the complementary strand of SEQ ID NO:2. Then, a polymerase chain reaction is performed on the sample using the aforementioned primer pair to obtain a product. Finally, the product is analyzed to detect the presence of non-tuberculous mycobacteria.

[0028] The sample can include specimens from various sources, such as one or a combination of blood, sputum, bronchoalveolar lavage fluid, urine, and feces. In some embodiments, the specimen provided in the method for detecting non-tuberculous mycobacteria contains non-tuberculous mycobacteria. In one embodiment, the non-tuberculous mycobacteria include, but are not limited to, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chelonae, Mycobacterium gordonae, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium terrae, Mycobacterium scrofulaceum, or Mycobacterium kansasii.

[0029] The selection of primer pairs is as described above and is not limited to SEQ ID NO:1 and SEQ ID NO:2 disclosed herein. In addition to the complementary strands of SEQ ID NO:1 and SEQ ID NO:2 that the primer pairs may include in the selection, the sequences shown in SEQ ID NO:1 and SEQ ID NO:2 may also allow for a certain degree of variation. That is to say, sequences with about 70% to about 99% homology to SEQ ID NO:1 and sequences with about 70% to about 99% homology to SEQ ID NO:2 also have the same efficacy when applied to this embodiment. For example, the selection of primer pairs may include the degenerate sequences of SEQ ID NO:1 and the degenerate sequences of SEQ ID NO:2. The "degenerate sequence" described herein refers to the substitution of some nucleotides in the oligonucleotide sequences disclosed herein with other nucleotides. In other words, the degenerate sequence of SEQ ID NO:1 means that, when the length of the SEQ ID NO:1 sequence remains unchanged, its oligonucleotides are allowed to have a variation degree of about 1% to about 30%. And the degenerate sequence of SEQ ID NO:2 means that, when the length of the SEQ ID NO:2 sequence remains unchanged, its oligonucleotides are allowed to have a variation degree of about 1% to about 30%. In some other embodiments, the selection of primer pairs may also include the derivative sequences of SEQ ID NO:1 and the derivative sequences of SEQ ID NO:2. The "derivative sequence" described herein refers to the modification that can be carried out at the 3'-end or 5'-end of the oligonucleotide sequences disclosed herein and still retains part or all of the sequences. In other words, the derivative sequence of SEQ ID NO:1 means that, when the length of the SEQ ID NO:1 sequence can be increased or decreased, its oligonucleotides are allowed to have a variation degree of about 1% to about 30%. And the derivative sequence of SEQ ID NO:2 means that, when the length of the SEQ ID NO:2 sequence can be increased or decreased, its oligonucleotides are allowed to have a variation degree of about 1% to about 30%. In some other embodiments, the primer pairs are selected from the group consisting of sequences with about 80% to about 99% homology to SEQ ID NO:1 (such as about 85%, about 90%, or about 95%) and sequences with about 80% to about 99% homology to SEQ ID NO:2 (such as about 85%, about 90%, or about 95%).

[0030] In some embodiments, the method for detecting non-tuberculous mycobacteria further includes providing at least one probe selected from the group consisting of SEQ ID NO:3, sequences having about 70% to about 99% homology with SEQ ID NO:3, the complementary strand of SEQ ID NO:3, SEQ ID NO:4, sequences having about 70% to about 99% homology with SEQ ID NO:4, and the complementary strand of SEQ ID NO:4. During detection, the probe can use only one sequence, or more than one sequence (such as two, three, four, etc.), and all can have similar detection effects.

[0031] The selection of the probe is as described above and is not limited to SEQ ID NOs: 3 and 4 disclosed herein. In addition to the complementary strands of SEQ ID NOs: 3 and 4, the sequences shown in SEQ ID NOs: 3 and 4 also allow a certain degree of variation. That is to say, sequences having about 70% to about 99% homology with SEQ ID NOs: 3 and 4 also have the same efficacy when applied to this embodiment. For example, the selection of the probe can include the degenerate sequence of SEQ ID NO:3. The degenerate sequence of SEQ ID NO:3 means that when the length of the SEQ ID NO:3 sequence remains unchanged, the oligonucleotide thereof can allow a variation degree of about 1% to about 30%. For example, the selection of the probe can include the degenerate sequence of SEQ ID NO:4. The degenerate sequence of SEQ ID NO:4 means that when the length of the SEQ ID NO:4 sequence remains unchanged, the oligonucleotide thereof can allow a variation degree of about 1% to about 30%. In some other embodiments, the selection of the probe can also include the derivative sequences of SEQ ID NOs: 3 and 4. For example, the derivative sequence of SEQ ID NO:3 means that when the length of the SEQ ID NO:3 sequence is increased or decreased at the 3' end or 5' end, the oligonucleotide thereof can allow a variation degree of about 1% to about 30%. For example, the derivative sequence of SEQ ID NO:4 means that when the length of the SEQ ID NO:4 sequence is increased or decreased at the 3' end or 5' end, the oligonucleotide thereof can allow a variation degree of about 1% to about 30%. In some other embodiments, the probe is selected from sequences having about 80% to about 99% homology with SEQ ID NO:3 (such as about 85%, about 90%, or about 95%).

[0032] In one embodiment, a polymerase chain reaction is performed on a sample using a primer pair and a probe to obtain a product, including performing the polymerase chain reaction such that the primer pair amplifies a partial sequence of the 16S ribosomal RNA gene sequence in the non-tuberculous mycobacteria group to obtain a product, wherein this partial sequence is SEQ ID NO:5 (Mycobacterium avium). The polymerase chain reaction is a molecular biology technique. A primer pair with oligonucleotide sequences is used to amplify a specific deoxyribonucleic acid (DNA) fragment. It should be understood that the sequences disclosed herein can be used in various polymerase chain reaction-based techniques. In one example, the polymerase chain reaction may include, but is not limited to, real-time quantitative polymerase chain reaction (real-time PCR). In one example, if the real-time polymerase chain reaction used is a probe-based fluorescence system, before performing the polymerase chain reaction on the sample using the primer pair to obtain a product, it further includes performing a hybridization reaction on the sample using the probe such that the probe binds to the target sequence. That is, the primer pair, the probe, and the sample are together subjected to a polymerase chain reaction to obtain a product.

[0033] An embodiment of the present disclosure also provides a kit for detecting non-tuberculous mycobacteria, including a primer pair. The above primer pair is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a sequence having about 70% to about 99% homology with SEQ ID NO:1, a sequence having about 70% to about 99% homology with SEQ ID NO:2, the complementary strand of SEQ ID NO:1, and the complementary strand of SEQ ID NO:2. In some embodiments, the primer pair is SEQ ID NO:1 and SEQ ID NO:2. In some embodiments, the primer pair is a sequence having about 70% to about 99% homology with SEQ ID NO:1 and a sequence having about 70% to about 99% homology with SEQ ID NO:2. In some embodiments, the primer pair is the complementary strand of SEQ ID NO:1 and the complementary strand of SEQ ID NO:2.

[0034] In certain embodiments, the kit for detecting non-tuberculous mycobacteria may further include a specimen. The source of the specimen can be one or a combination of blood, sputum, bronchoalveolar lavage fluid, urine, and feces. For example, this kit for detecting non-tuberculous mycobacteria can be applied to various medical units to detect by collecting body fluids or excreta of an individual (such as a human).

[0035] In some embodiments, the kit for detecting non-tuberculous mycobacteria may further comprise a target gene, and this target gene refers to the 16s ribosomal RNA gene sequence of non-tuberculous mycobacteria. Furthermore, in some embodiments, the kit for detecting non-tuberculous mycobacteria may further comprise a template with a length of about 100 base pairs to about 250 base pairs. For example, this template may be a partial sequence in the 16s ribosomal RNA sequence, such as the sequence shown in SEQ ID NO:5, which has a length of 140 base pairs. However, in some other embodiments, the template does not contain the sequence shown in SEQ ID NO:5 and is a synthetic sequence with a length of about 100 base pairs to about 250 base pairs, which can also bind to the primer pair in this embodiment and thus be amplified. In some embodiments, the sequence shown in SEQ ID NO:5 can be directly constructed into different vectors, and when the vector carrying SEQ ID NO:5 is used as a template for amplification, the specificity is high and the detection efficiency is excellent.

[0036] In some embodiments, the kit for detecting non-tuberculous mycobacteria may further comprise at least one probe selected from the group consisting of SEQ ID NO:3, a sequence having about 70% to about 99% homology with SEQ ID NO:3, the complementary strand of SEQ ID NO:3, SEQ ID NO:4, a sequence having about 70% to about 99% homology with SEQ ID NO:4, and the complementary strand of SEQ ID NO:4. In some embodiments, the probe is SEQ ID NO:3, SEQ ID NO:4, or a combination thereof.

[0037] To further confirm that various embodiments of the present invention can be used to detect the presence of mycobacteria, the following tests were conducted. It should be noted that the following examples are provided for illustrative purposes only and do not limit the present invention.

[0038] Primer and probe design

[0039] The 16s ribosomal RNA gene sequence of the non-tuberculous mycobacteria group is highly conserved. Therefore, in this experiment, online design programs such as primer 3 and GenScript Real-time PCR Primer Design were used to design primers and probes for the 16s ribosomal RNA sequences of 9 non-tuberculous mycobacteria (Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chelonae, Mycobacterium gordonae, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium terrae, Mycobacterium scrofulaceum, or Mycobacterium kansasii) and 1 Mycobacterium tuberculosis.

[0040] According to the information provided by the GenBank database, Figure 1Shows a partial sense strand sequence of the 16S ribosomal RNA gene sequence. In this experiment, the primer pair is SEQ ID NO:1 and SEQ ID NO:2. The nucleotide sequence shown in SEQ ID NO:1 is designed for the position of the 151-174th base pairs (as shown by the solid box with a right arrow). The nucleotide sequence shown in SEQ ID NO:2 is designed for the position of the 271-290th base pairs (as shown by the solid box with a left arrow). The probes are SEQ ID NOs:3 and 4. The nucleotide sequence shown in SEQ ID NO:3 is designed for the fragment between the 181-194th base pairs (as shown by the dashed box with a right arrow). The nucleotide sequence shown in SEQ ID NO:4 is designed for the fragment between the 211-223rd base pairs (as shown by the dashed box with a left arrow). Accordingly, the product amplified by the primer pair SEQ ID NOs:1 and 2 has a length of 140 base pairs.

[0041] Specifically, the 5th-11th, 13th, and 15th nucleotides of the forward primer SEQ ID NO:1 are BYBDSDRKS, where B represents that the nucleotide can be selected from g, c, or t (i.e., not a, and the synthesis ratio is about 33% each), Y represents that the nucleotide can be selected from c or t (the synthesis ratio is about 50% each), D represents that the nucleotide can be selected from a, g, or t (i.e., not c, and the synthesis ratio is about 33% each), S represents that the nucleotide can be selected from g or c (the synthesis ratio is about 50% each), R represents g or a (the synthesis ratio is about 50% each), and K represents g or t (the synthesis ratio is about 50% each). When preparing the forward primer SEQ ID NO:1, all the above nucleotide combinations are mixed in.

[0042] Sensitivity analysis of the primer pair

[0043] According to the 16S ribosomal RNA gene sequence shown in the aforementioned GenBank data, 9 non-tuberculous mycobacteria (Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chelonae, Mycobacterium gordonae, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium terrae, Mycobacterium scrofulaceum, and Mycobacterium kansasii) were respectively cloned into the pJET1.2 / blunt vector (Protech CO., Ltd, GenBank: Y14837.1) to obtain 9 standard plasmids carrying the 16S ribosomal RNA gene sequence (hereinafter simply referred to as 16S rRNA standard plasmids).

[0044] Prepare a reaction mixture containing a template (16s rRNA standard plasmid), polymerase chain reaction reagents (QuantiNova probe master mix), a forward primer (SEQ ID NO:1) with a concentration of 200 nM, and a reverse primer (SEQ ID NO:2) with a concentration of 300 nM. The polymerase chain reaction conditions are 2 minutes at 95°C, denaturation at 95°C for 5 seconds, annealing / amplification at 60°C for 5 seconds, and 45 cycles of reaction are carried out.

[0045] Please refer to Figure 2 , according to some embodiments of the present disclosure, a polymerase chain reaction is performed with the primer pair SEQ ID NO:1 and SEQ ID NO:2 to detect clinical specimens of Mycobacterium tuberculosis group and non-tuberculous mycobacteria. From left to right, columns 2-6 are Mycobacterium tuberculosis (TB), columns 7-15 and 16-19 are non-tuberculous mycobacteria (Mycobacterium avium, Mycobacterium gordonae, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium scrofulaceum, Mycobacterium terrae, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium fortuitum, and Mycobacterium fortuitum), columns 1, 16, and 21 are molecular weight marker columns (marker ladder), and column 20 is a no-template control (NTC). The electrophoresis result diagram shows that only non-tuberculous mycobacteria have a specific amplification product (140 bp), and no amplification product is produced in the Mycobacterium tuberculosis group. Therefore, the primer pair SEQ ID NOs:1, 2 can accurately distinguish non-tuberculous mycobacteria from the Mycobacterium tuberculosis group.

[0046] Sensitivity analysis of primer pairs and probes

[0047] According to the operation manual of the commercially available real-time quantitative polymerase chain reaction kit (QuantiNova Probe PCR Kit, Qiagen), the reaction mixture contains a template (16s rRNA standard plasmid), 11 μL of real-time quantitative polymerase chain reaction reagent (QuantiNova probe master mix), a forward primer (SEQ ID NO:1) with a concentration of 200 nM, a reverse primer (SEQ ID NO:2) with a concentration of 300 nM, a probe 1-1 (SEQ ID NO:3) with a concentration of 200 nM, and a probe 1-2 (SEQ ID NO:4) with a concentration of 200 nM, and is formulated into a reaction mixture with a total volume of 25 μL. The real-time quantitative polymerase chain reaction conditions are denaturation at 95°C for 5 seconds, annealing / amplification at 60°C for 5 seconds, and the reaction mixture is subjected to 45 cycles of reaction in a real-time quantitative polymerase chain reaction instrument (CFX-96, BioRad).

[0048] It should be noted that in this experiment, real-time quantitative polymerase chain reaction was carried out with different template amounts to test the sensitivity of the primer pair SEQ ID NO:1 and SEQ ID NO:2. According to the formula of the aforementioned reaction mixture, 7 reaction mixtures with different template amounts were prepared, each containing 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 and 10 7 copy numbers of the 16srRNA standard plasmid. Refer to Figure 3 (the 16s rRNA standard plasmid of Mycobacterium avium), which are the amplification curve graph and standard curve graph obtained after real-time quantitative polymerase chain reaction respectively. As Figure 3 shown, the horizontal axis is the number of reaction cycles (cycles), and the vertical axis is the fluorescence intensity (ΔRn). It can be seen from this amplification curve graph that the fluorescence values of 10 to 10 7 copy numbers all show an upward positive trend. According to the threshold cycle (Ct) values obtained from different copy numbers, the detection range of the primer pair and probe set designed in the present disclosure can be verified. Please refer to Figures 4 to 12 , which shows the R 7 values obtained by performing real-time quantitative polymerase chain reaction on 9 non-tuberculous mycobacteria under different template amounts (10 - 10 2 copy numbers) according to some embodiments of the present disclosure. The R 2The values are all greater than 0.94, indicating a high accuracy of this regression model. In addition, please refer to Table 1-9 below. When detecting 5-80 copies of 9 non-tuberculous mycobacteria, the lowest detection limit can reach 5 copies per reaction, and all 6 detections have detected them, with a detection rate of 100%.

[0049] Table 1, Mycobacterium avium

[0050]

[0051]

[0052] Table 2, Mycobacterium intracellulare

[0053] Copy number Threshold cycle (Ct) Detection rate 80 34.2 6 / 6 40 36.4 6 / 6 20 38.3 6 / 6 10 39.5 6 / 6 5 40.5 6 / 6

[0054] Table 3, Mycobacterium chelonae

[0055] Copy number Threshold cycle (Ct) Detection rate 80 36.5 6 / 6 40 37.8 6 / 6 20 38.2 6 / 6 10 38.1 6 / 6 5 38.6 6 / 6

[0056] Table 4, Mycobacterium gordonae

[0057] Copy number Threshold cycle (Ct) Detection rate 80 34.0 6 / 6 40 34.6 6 / 6 20 34.8 6 / 6 10 34.9 6 / 6 5 35.0 6 / 6

[0058] Table 5, Mycobacterium abscessus

[0059]

[0060]

[0061] Table 6, Mycobacterium fortuitum

[0062] Copy number Threshold cycle (Ct) Detection rate 80 34.4 6 / 6 40 36.4 6 / 6 20 37.8 6 / 6 10 38.4 6 / 6 5 40.7 6 / 6

[0063] Table 7, Mycobacterium terrae

[0064] Copy number Threshold cycle (Ct) Detection rate 80 37.1 6 / 6 40 37.3 6 / 6 20 37.2 6 / 6 10 37.6 6 / 6 5 37.6 6 / 6

[0065] Table 8, Mycobacterium scrofulaceum

[0066] Copy number Threshold cycle (Ct) Detection rate 80 33.8 6 / 6 40 34.1 6 / 6 20 34.3 6 / 6 10 34.5 6 / 6 5 34.7 6 / 6

[0067] Table 9, Mycobacterium kansasii

[0068] Copy number Threshold cycle (Ct) Detection rate 80 37.7 6 / 6 40 39.3 6 / 6 20 40.4 6 / 6 10 40.8 6 / 6 5 41.7 6 / 6

[0069] Clinical tests

[0070] Detect clinical specimens (as shown in Table 10 below) with the primer pair (SEQ ID NOs: 1-2) and probe set (SEQ ID NOs: 3-4) of the present disclosure, and use an ABI Step One thermal cycler to detect 82 clinical samples.

[0071] Table 10, Number of Positive Clinical Specimens of Each Mycobacterium

[0072]

[0073] For the results, please refer to Figure 13 As shown, in the amplification curve of real-time quantitative polymerase chain reaction for 82 clinical samples, Mycobacterium tuberculosis group (TB) and nontuberculous mycobacteria (NTM) can be accurately identified, and there is no amplification reaction in the specimens of the nontuberculous mycobacteria group.

[0074] The foregoing has outlined the features of several embodiments so that those skilled in the art may better understand aspects of the present disclosure. Those skilled in the art should understand that the present disclosure can be readily used as a basis for designing or modifying other processes and structures for achieving the same purposes and / or achieving the same advantages as the embodiments introduced herein. Those skilled in the art should also recognize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of the present disclosure. Sequence Listing <110> Delta Electronics, Inc. Pei-Chen Tsai <120> Method for Detecting Nontuberculous Mycobacteria, Primer Set, and Kit Thereof <130> NP-26367-TW <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 1 catgbybdsd rgkgsaaagc tttt 24 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 2 gccgtatctc agtcccagtg 20 <210> 3 <211> 14 <212> Artificial Sequence <220> <223> Probe 1-1 <400> 3 tgggatgggc ccgc 14 <210> 4 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Probe 1-2 <400> 4 ccattacccc accaac 16 <210> 5 <211> 140 <212> DNA <213> Mycobacterium avium <400> 5 catgtcttct ggtggaaagc ttttgcggtg tgggatgggc ccgcggccta tcagcttgtt 60 ggtggggtga cggcctacca aggcgacgac gggtagccgg cctgagaggg tgtccggcca 120 cactgggact gagatacggc 140

Claims

1. Use of a primer pair and a probe pair in the preparation of a kit for detecting non - tuberculous mycobacteria, wherein the primer pair and the probe pair are selected from: (1) The primer pair of SEQ ID NO:1 and SEQ ID NO:2 and the probe pair of SEQ ID NO:3 and SEQ ID NO:4, or (2) The primer pair of SEQ ID NO:1 and SEQ ID NO:2 and the probe pair of the complementary strand of SEQ ID NO:3 and the complementary strand of SEQ ID NO:

4.

2. The use according to claim 1, wherein the detection comprises the following steps: Providing a sample; Providing a primer pair and a probe pair; Performing a polymerase chain reaction on the sample with the primer pair and the probe pair to obtain a product; and Analyzing the product to detect the presence of non - tuberculous mycobacteria.

3. The use according to claim 2, wherein the step of providing the sample comprises providing a specimen containing non - tuberculous mycobacteria.

4. The use according to claim 3, wherein the specimen is one or a combination of blood, sputum, bronchoalveolar lavage fluid, urine, and feces.

5. The use according to claim 3, wherein the step of performing the polymerase chain reaction on the sample with the primer pair and the probe pair to obtain the product comprises performing a polymerase chain reaction such that the primer pair amplifies a partial sequence of the 16S ribosomal RNA gene sequence in the non - tuberculous mycobacteria to obtain the product, wherein the partial sequence is SEQ ID NO:

5.

6. The use according to claim 1, wherein the step of performing the polymerase chain reaction on the sample with the primer pair, the probe pair to obtain the product, the polymerase chain reaction is a real - time quantitative polymerase chain reaction.

7. A kit for detecting non - tuberculous mycobacteria, comprising a primer pair and a probe pair, which are selected from: (1) The primer pair of SEQ ID NO:1 and SEQ ID NO:2 and the probe pair of SEQ ID NO:3 and SEQ ID NO:4, or (2) The primer pair of SEQ ID NO:1 and SEQ ID NO:2 and the probe pair of the complementary strand of SEQ ID NO:3 and the complementary strand of SEQ ID NO:

4.

8. The kit for detecting non - tuberculous mycobacteria according to claim 7, further comprising a specimen, wherein the specimen is one or a combination of blood, sputum, bronchoalveolar lavage fluid, urine, and feces.

9. The kit for detecting non-tuberculous mycobacteria according to claim 7 further comprises a target gene, wherein the target gene is the 16S ribosomal RNA sequence of non-tuberculous mycobacteria.

10. The kit for detecting non-tuberculous mycobacteria according to claim 7 further comprises a template.

11. The kit for detecting non-tuberculous mycobacteria according to claim 10, wherein the template is SEQ ID NO:

5.

12. A combination of a primer set and a probe set, comprising: (1) A primer pair of SEQ ID NO:1 and SEQ ID NO:2 and a probe pair of SEQ ID NO:3 and SEQ ID NO:4 or (2) A primer pair of SEQ ID NO:1 and SEQ ID NO:2 and a probe pair of the complementary strand of SEQ ID NO:3 and the complementary strand of SEQ ID NO:4.

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