Patent Publication Number: US-2021164058-A1

Title: Primer set for use in detection of candida auris, candida auris detection kit, and candida auris detection method

Description:
TECHNICAL FIELD 
     The present invention relates to a primer set for use in the detection of  Candida auris , a  Candida auris  detection kit, and a  Candida auris  detection method. 
     BACKGROUND ART 
       Candida auris  is a pathogenic species of yeast which was first discovered in Tokyo in 2005 and was reported by the first present inventor as a new species (Non-Patent Literature 1). 
     Until August 2017,  Candida auris  discovered in Japan was found in a culture of a topically infected site (external ear canal) and has relatively low pathogenicity.  Candida auris  was just a single strain that was not drug-resistant, although  Candida auris  exerted slightly low resistivity to some of anti-fungal drugs (e.g., fluconazole). 
     After that time, however, virulence-increased multidrug-resistant strains of  Candida auris  which can cause sepsis and the like have been reported with the spreading of  Candida auris  all over the world including South Korea, India, North Africa, Venezuela, the United Kingdom and the United States. By 2016,  Candida auris  has been recognized as a world&#39;s first fungus that can cause global outbreak (pandemic) (Non-Patent Literature 2). In the latest report published in May 17, 2017, it was reported that 122 persons (7 persons in the last year) were infected with  Candida auris  in the United States and  Candida auris  had high mortality. In the United Kingdom,  Candida auris  has also been spreading, the number of infected persons already reaches around 200 persons,  Candida auris  is reported as “Japanese Fungus” by the media, and alert has been issued to medical/healthcare institutions. 
     Under the current circumstances, the infection by  Candida auris  is limited to compromised persons, and the outbreak of the infection occurs in the form of hospital-associated infection. From the view of the overseas spreading situations, it is considered that there is a high possibility that the hospital-associated infection is spread through nasal cavities, external ear canals and so on with a normal persons acting as a vector, as in the first case reported firstly by the first present inventor. Actually in the United States which has already been under the spread of the infection by the fungus, it is believed that the infection by the fungus is spread easily thorough skin such as fingers of a healthcare professional (Non-Patent Literature 3). Accordingly, there is a high possibility that a fungus that has acquired increased virulence and drug-resistivity overseas is imported into our country because of flourishing international exchanges and the opening of Tokyo Olympic Games, and it has been urgently demanded to prepare testing systems for preventing the import/export of toxic fungus before it occurs. 
     CITATION LIST 
     Non-Patent Literature 
     
         
         Non-Patent Literature 1: Satoh K, Makimura K, Hasumi Y, Nishiyama Y, Uchida K, Yamaguchi H. 2009.  Candida auris  sp. nov., a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a Japanese hospital. Microbiol Immunol 53:41-44. https://doi.org/10.1111/j.1348-0421.2008.00083.x. 
         Non-Patent Literature 2: Website of the US Centers for Disease Control and Prevention (CDC), Tracking  Candida auris  https://www.cdc.gov/fungal/diseases/candidiasis/tracking-c-auris.html (connected on Apr. 22, 2018) 
         Non-Patent Literature 3: Shawn R. Lockhart, Elizabeth L. Berkow, Nancy Chow, Rory M. Welsh,  Candida auris  for the Clinical Microbiology Laboratory: Not Your Grandfather&#39;s  Candida  Species, Clinical Microbiology Newsletter, Volume 39, Issue 13, 2017, Pages 99-103, ISSN 0196-4399, https://doi.org/10.1016/j.clinmicnews.2017.06.003. (http://www.sciencedirect.com/science/article/pii/S0196439917300417) 
         Non-Patent Literature 4: Nakayama T, Yamazaki T, Yo A, Tone K, Mahdi Alshahni M, Fujisaki R, Makimura K. 2017. Detection of fungi from an indoor environment using loop-mediated isothermal amplification (LAMP) method. Biocontrol Sci 22:97-104. https://doi.org/10.4265/bio.22.97. 
         Non-Patent Literature 5: Shigekazu Iguchi, Ryo Mizushima, Keisuke Kamada, Yasutomo Itakura, Atsushi Yoshida, Yutaka Uzawa, Yuko Arai, Miyako Takaoka, SumieSato, Aeko Goto, Toshiko Karasawa, Naoki Tsuruoka, Daisuke Totsuka, Erika Ono, Manabu Nonaka, Koichi Makimura, Ken Kikuchi: The Second  Candida auris  Isolate from Aural Discharge in Japan. Jpn J Infect Dis. 2018 Mar. 22; 71(2):174-175. doi: 10.7883/yoken.JJID.2017.466. Epub 2018 Feb. 28. 
       
    
     SUMMARY OF INVENTION 
     Technical Problem 
     However,  Candida auris  is a new species of pathogenic fungus (yeast), and cannot be discriminated from closely related species thereof (e.g.,  Candida haemulonii, Rhodotorula glutinis ) by an automated identification system that has been commonly used in a clinical laboratory, such as VITEK2 YST card (bioMerieux, Maracy I&#39;Etoile, France) and API20C AUX (bioMerieux). Accordingly, the type of the species of the fungi cannot be determined accurately. For these reasons, the establishment of a method for detecting (identifying)  Candida auris  with high sensitivity and in a specific manner is a major problem in diagnosis, treatment, clinical tests, control of hospital-associated infections, and epidemiologic analysis. Moreover, in order to make a treatment for  Candida auris  infection effective and prevent the spread of the infection, rapid detection ability as well as high detection accuracy are absolutely necessary. 
     The present invention has been made under these circumstances, and an object of the present invention is to provide a detection method which can detect  Candida auris  rapidly and accurately. Another object of the present invention is to provide a primer set and a detection kit which can be used in the detection method. 
     Solution to Problem 
     In order to solve the problem, the primer set for use in the detection of  Candida auris  according to the present invention is a primer set for use in the detection of  Candida auris , which includes four types of primers consisting of FIP, BIP, F3 and B3, and is intended to be used in the detection of  Candida auris  by amplifying a  Candida auris  target sequence in a specimen by the LAMP method, 
     the primer set being characterized in that: 
     FIP is a polynucleotide including 5 to 20 nucleotides located on the 5′-terminal side and 5 to 20 nucleotides located on the 3′-terminal side in the nucleotide sequence represented by SEQ ID NO: 1; 
     BIP is a polynucleotide including 5 to 20 nucleotides located on the 5′-terminal side and 5 to 20 nucleotides located on the 3′-terminal side in the nucleotide sequence represented by SEQ ID NO: 2; 
     F3 is a polynucleotide including SEQ ID NO: 3; and 
     B3 is a polynucleotide including SEQ ID NO: 4. 
     In the primer set for use in the detection of  Candida auris , it is preferred that FIP is a polynucleotide including SEQ ID NO: 1 and BIP is a polynucleotide including SEQ ID NO: 2. 
     In the primer set for use in the detection of  Candida auris , it is more preferred that polynucleotides respectively including SEQ ID Nos: 5 and 6 are further included as loop primers. 
     The  Candida auris  detection kit according to the present invention includes the primer set for use in the detection of  Candida auris.    
     The method for detecting  Candida auris  according to the present invention is a method for detecting  Candida auris  including subjecting a nucleic acid sample obtained from a specimen to a nucleic acid amplification reaction by the LAMP method and detecting an amplification product, 
     the method being characterized in that: 
     a primer set to be used in the LAMP method includes four types of primers consisting of FIP, BIP, F3 and B3; 
     FIP is a polynucleotide including 5 to 20 nucleotides located on the 5′-terminal side and 5 to 20 nucleotides located on the 3′-terminal side in the nucleotide sequence represented by SEQ ID NO: 1; 
     BIP is a polynucleotide including 5 to 20 nucleotides located on the 5′-terminal side and 5 to 20 nucleotides located on the 3′-terminal side in, the nucleotide sequence represented by SEQ ID NO: 2; 
     F3 is a polynucleotide including SEQ ID NO: 3; and 
     B3 is a polynucleotide including SEQ ID NO: 4. 
     In the method for detecting  Candida auris , it is preferred that FIP is a polynucleotide including SEQ ID NO: 1 and BIP is a polynucleotide including SEQ ID NO: 2. 
     In the method for detecting  Candida auris , it is more preferred that the primer set further includes polynucleotides respectively including SEQ ID Nos: 5 and 6 as loop primers. 
     Advantageous Effects of Invention 
     According to the method for detecting  Candida auris  of the present invention, it is possible to detect  Candida auris  rapidly and accurately. The primers and the detection kit can be used in the method for detecting  Candida auris  of the present invention. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a diagram showing the results of the detection of amplification products by the detection method of the present invention using the LAMP Auris primer set using pTAC-2 Auris plasmid carrying a DNA fragment specific to  Candida auris , in which the results are expressed in terms of the number of copies per detection reaction (copies/reaction). A: negative control (no reaction), B: 2×10 0  copies/reaction (34 min), C: 2×10 1  copies/reaction (33 min), D: 2×10 2  copies/reaction (26 min), E: 2×10 3  copies/reaction (24 min), F: 2×10 4  copies/reaction (24 min), G: 2×10 6  copies/reaction (22 min), H: 2×10 8  copies/reaction (17 min). 
         FIG. 2  is a diagram showing the results of the detection of  Candida auris  ( C. auris ) in a clinical specimen by the detection method of the present invention. 
         FIG. 3  is a diagram showing the results of the detection of  Candida auris  ( C. auris ) in an environmental sample by the detection method of the present invention. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     The present inventors have focused attention on the LAMP method as a means for detecting  Candida auris  rapidly and specifically and with high sensitivity. The present inventors have succeeded in the designing of four types of primers for amplifying target DNA of  Candida auris  specifically by the LAMP method and two types of loop primers for accelerating gene amplification. As a result, the present invention has been accomplished. 
     The LAMP method is known conventionally, and is a method in which amplification is performed by utilizing a strand displacement reaction using four types of primers which are combinations of six regions selected in the sequence for a target gene. 
     The designing of the primers in the LAMP method is carried out by utilizing six regions, i.e., F3 region, F2 region, F1 region, B1 region, B2 region and B3 region as observed from the 5′-side, in a region to be amplified (wherein the region is also referred to as “template (nucleotide, DNA)”, hereinafter). Regions respectively complementary to these six regions are referred to as “F3c region”, “F2c region”, “F1c region”, “B1c region”, “B2c region” and “B3c region”. In the LAMP method, four types of primers (two types of Inner primers (FIP and BIP), and two types of Outer primers (F3 primer and B3 primer)) are used. The Inner primers are respectively formed by linking F1c region (i.e., a region complementary to F1 region) to F2 region and linking B1c (i.e., a region complementary to B1 region) to B2 region. In general, the LAMP method has such characteristics that a denaturation reaction from a single strand to a double strand is not needed and the reaction can proceed at a constant temperature ranging from 60 to 65° C. when compared with PCR method, and also has such characteristics that an apparatus such as a thermal cycler is not required, the amplification speed is rapid, and the specificity is also high. 
     The LAMP method using loop primers is also known conventionally. In general, each of the loop primers is designed as a primer having a sequence complementary to a single strand region (a region between B1 region and B2 region, or a region between F1 region and F2 region) of a 5′-terminal-side loop of a dumb-bell structure in an amplification product of the LAMP method. The loop primers are called as “loop primers B(LB)” and “loop primers F(LF)”, respectively. When the loop primers are used, the number of origins of DNA synthesis can be increased and therefore gene amplification can be accelerated. 
     In the method for detecting  Candida auris  of the present invention, as a specimen to be detected, a material such as a clinical specimen collected from a subject (e.g., blood, a tissue, ascitic fluid, a bronchoalveolar lavage fluid, and a skin, mucosal or external ear canal swab) or cultured cells can be used. These specimens may be subjected to the concentration or separation of cells, the isolation or concentration of a nucleic acid from cells, or the like as a pretreatment for an amplification reaction by the LAMP method. 
     A sample nucleic acid can be prepared in accordance with a known method from a specimen to be detected. As the method for preparing the sample nucleic acid, a conventional method can be employed, and examples of the method include a chemical lysis (e.g., Proteinase K) treatment, a physical disruption (e.g., bead disruption) treatment, an alkaline lysis method, and a method of purifying a nucleic acid by the extraction with phenol/chloroform or using magnetic beads, a silica membrane or the like (Non-Patent Literature 4). Alternatively, a commercially available nucleic acid extraction kit (e.g., Kaneka Easy DNA Extraction kit version 2 manufactured by Kaneka Corporation) may be used as required. 
     The primer set to be used in the detection method of the present invention (i.e., the primer set for use in the detection of  Candida auris ) includes four types of primers (FIP, BIP, F3, B3), and may also include loop primers (Loop-B, Loop-F) as required. 
     The primers (FIP, BIP, F3, B3) and the loop primers (Loop-B, Loop-F) include sequences respectively including the polynucleotides shown below. The primers (FIP, BIP, F3, B3) and the loop primers (Loop-B, Loop-F) are designed from the findings of the present inventors on the basis of a nucleotide sequence (target sequence) specific to  Candida auris  and suitable for the LAMP method. 
     A preferred embodiment of the primer sets of the present invention is shown below. 
     
       
         
           
               
            
               
                 (Primer set for use in the detection of  Candida   
               
               
                   auris ) 
               
               
                 FIP: 
               
               
                 (SEQ ID NO: 1) 
               
               
                 AGGCTACTGAGCTTGCTGGTGTAACCAAACCAACAGGAGAGG 
               
               
                   
               
               
                 BIP: 
               
               
                 (SEQ ID NO: 2) 
               
               
                 ACGGTTTCAGGGTTAGCATGGCTCAACAAAGTCGCTGGTACA 
               
               
                   
               
               
                 F3: 
               
               
                 (SEQ ID NO: 3) 
               
               
                 GGGAAAGGAACCCTGACCT 
               
               
                   
               
               
                 B3: 
               
               
                 (SEQ ID NO: 4) 
               
               
                 GGACACAGCATTCGAAGTGT 
               
               
                   
               
               
                 Loop-F: 
               
               
                 (SEQ ID NO: 5) 
               
               
                 CATCTCGAAGGCCTCGGT 
               
               
                   
               
               
                 Loop-B: 
               
               
                 (SEQ ID NO: 6) 
               
               
                 CACATACTCGAACGGAGTC 
               
            
           
         
       
     
     The primer set of the present invention may include those a primer in which a design change, such as the substitution or deletion of a nucleotide in the sequence or the change in the length of the sequence, is made as long as the specific detection of  Candida auris  by the LAMP method cannot be inhibited. Particularly with respect to the sequence for each of FIP primer and BIP primer, about 10 nucleotides located at the center part of the primer may be an arbitrary sequence, or an arbitrary sequence composed of about 1 nucleotide to 200 nucleotides may be inserted at the center part. With respect to Loop-F primer and LoopB primer, any primers can act as the Loop-F primer and the Loop-B primer, as long as each of the primers includes a sequence that is complementary to a single-strand domain (located between B1 region and B2 region or between F1 region and F2 region) in a 5′-terminal-side loop of a dumb-bell structure of an amplification product of the LAMP method and is specific to the domain. Therefore, the sequences for the Loop-F primer and the Loop-B primer are not limited to the sequences represented by SEQ ID NO: 5 and SEQ ID NO: 6. 
     Namely, in the primer set of the present invention, FIP is required to be a polynucleotide which includes 5 to 20 nucleotides located on the 5′-terminal side and 5 to 20 nucleotides located on the 3′-terminal side in the nucleotide sequence represented by SEQ ID NO: 1, and BIP is required to be a polynucleotide which includes 5 to 20 nucleotides located on the 5′-terminal side and 5 to 20 nucleotides located on the 3′-terminal side in the nucleotide sequence represented by SEQ ID NO: 2. 
     In the detection method of the present invention, the nucleic acid sample in the specimen is subjected to a nucleic acid amplification reaction by the LAMP method using the primers (FIP, BIP, F3, B3) and optionally using the loop primers (Loop-B, Loop-F), and an amplification product is then detected. 
     The conditions for the amplification of the target sequence in the nucleic acid sample by the LAMP method are not particularly limited, and may be adjusted appropriately. A specific example of the temperature to be employed for the nucleic acid amplification reaction by the LAMP method is a temperature falling within the range from about 50° C. to about 65° C., and a specific example of the reaction time to be employed is a time period falling within the range from 20 minutes to 60 minutes. 
     In addition to the above-mentioned primer set, the  Candida auris  detection kit to be used for the detection method of the present invention can also include a polymerase, a buffer, dNTPs, MgSO 4  and the like as reagents for use in the amplification of the sample nucleic acid. The concentrations of the reagents to be used in the detection method of the present invention and the like can be adjusted appropriately depending on the volumes of the reagents, the reaction time and the like. As the polymerase to be used in the amplification of the sample nucleic acid, a known polymerase can be used appropriately. For example, Bst DNA Polymerase (manufactured by Eiken Chemical Co., Ltd.), Csa DNA Polymerase (manufactured by Nippon Gene Co., Ltd.) and the like can be used. 
     In the detection kit, the primer set and other regents may be included separately, or some of them may be prepared in the form of a mixture. The primer set consists of four types of primers and loop primers, and these primers may be included separately or some of them may be prepared in the form of a mixture. 
     Furthermore, in the detection method of the present invention, a known method may be employed appropriately for the detection of an amplification product by the LAMP method. For example, for the detection of the amplification product, a method in which the occurrence of white turbidity of a reaction is observed with naked eyes, a method in which the white turbidity of a reaction solution is measured using a spectrophotometer, and a method in which a fluorescence is detected visually, a method in which a labeled oligonucleotide or a fluorescent intercalator capable of recognizing an amplified nucleotide sequence specifically is used can be exemplified. Alternatively, it is also possible that a reaction solution obtained after the completion of the reaction is subjected to agarose gel electrophoresis without any modification, then the gel is stained with ethidium bromide and then a ladder-like electrophoresis image specific to an amplification product is confirmed by the irradiation with ultraviolet ray. 
     In the detection method of the present invention, the detection of an amplification product obtained by the LAMP method may include the detection of the presence or absence of the amplification product as well as the measurement of the quantity of the amplification product. 
     In the detection method of the present invention, the nucleic acid sample in the specimen is subjected to a nucleic acid amplification reaction by the LAMP method using the detection kit including the primer set, and an amplification product is then detected. The primer set is designed employing a nucleotide sequence specific to  Candida auris  as a target. 
     In the detection method of the present invention,  Candida auris  can be detected specifically with high sensitivity without detecting a closely related species thereof. Therefore,  Candida auris  can be detected accurately even when a specimen contaminated with various microorganisms (e.g., a contaminated clinical specimen, an environmental specimen) is used. Furthermore, in the detection method of the present invention, the time required for the detection is short (about 20 minutes to 60 minutes) and therefore the detection can be achieved rapidly. 
     The  Candida auris  detection method, the primer set and the detection kit of the present invention are not limited to the above-mentioned embodiments. 
     EXAMPLES 
     Hereinbelow, the present invention will be described specifically by way of examples. However, the present invention is not limited in any way by these examples. 
     &lt;Example 1&gt; Designing of LAMP Auris Primer Set 
     In order to design a primer set to be used in the LAMP method, the genome sequences for four types of  Candida  species, i.e.,  C. auris  (PRJNA342691),  C. tropicalis  (GCF_000006335.2),  C. albicans  (GCA_000182965.3) and  C. lusitaniae  (LYUB00000000.2), were aligned and were compared with one another using Mauve (version 20150226), and a sequence having low homology with other  Candida  species was searched for. As a result, about several hundreds nucleotide sequences were obtained. 
     Most of the several hundreds nucleotide sequences were not suitable for the designing of primers. On the basis of the technical knowledge based on the experience of the present inventors, four types of nucleotide sequences (candidate sequences) that were assumed to be used for the designing of the LAMP primer set were selected (SEQ ID NOs: 7 to 10). 
     
       
         
           
               
            
               
                 (SEQ ID NO: 7) 
               
               
                 CACTACAGCAGGATCAACGGATGCTTCATACTCTGAAATCACCTTTAATG 
               
               
                   
               
               
                 CTGGGATTGGCGCCCACACAAAGTTGGCTGGGTGGACAAACTCCTCCACA 
               
               
                   
               
               
                 GAAACAGAACCGAAACGGCCAGCGAGGAACAACGAAGCAGCAACGTCAGC 
               
               
                   
               
               
                 CTTCAAGGTTGAGCCAGCATCCGAAGATACCACAACAACCTTGCGTGCAG 
               
               
                   
               
               
                 ACGAAGGCACAGAAGCCACGAAGTGTTCTTGAGCAAATGGGAAAGGAACC 
               
               
                   
               
               
                 CTGACCTTAACCAAACCAACAGGAGAGGAAACCGAGGCCTTGGAGATGAC 
               
               
                   
               
               
                 ACCAGCAAGCTCAGTAGCCTGGTGAGCACCAAAAGCGACAAAAACAGTTT 
               
               
                   
               
               
                 CAGGGTTAGCATGGCCCACATACTCGAACGGAGTCAAATTTGTACCAGCG 
               
               
                   
               
               
                 ACTTTGTTGAACACTTCGAATGCTGTGTCCACTGCCTTTTCCAAAGAATA 
               
               
                   
               
               
                 GTCGCCGTCGGGAATCGAAGACACCAATTGCTGGTATAAGCGGCCCACAT 
               
               
                   
               
               
                 CGGTCACAGACAAGATGTCATCGAACTTGGAGATCGCTTTAGCAAACTCA 
               
               
                   
               
               
                 GGACCGTCGAAAACGTGCAAGGCTGGTCCCTGAAGCAAAGTAGCCACAAA 
               
               
                   
               
               
                 GTGAGTGAAGATGGTGATGTACTGCAACTCCACGGCGCTCTGCGAGTCCG 
               
               
                   
               
               
                 CCGGGGCCACCACAGGGATACCCGTGGAGCGAGCAGTTGCCAAAGGAGTC 
               
               
                   
               
               
                 GTATAGTTTGAAACCAACGAGTTTGTTTCCACATCAAAGTCGATAGCGGA 
               
               
                   
               
               
                 AACGTTCAAGGTCAATGGCAATTTTGCAGCCTTGGCCAAAGTGGGCTGCA 
               
               
                   
               
               
                 TCCATGGCAAGGCGTTGGCGCCCAACACAGCAGTATGTGGTCCAGTGGAG 
               
               
                   
               
               
                 GACACGTTGCTAGCGGCATAGC 
               
               
                   
               
               
                 (SEQ ID NO: 8) 
               
               
                 AGTAAGAGCTGCGGTCATCAAGCTCAACATCTTCATCGTCTATCGCCGAC 
               
               
                   
               
               
                 AGAGACGCCTCCATTTGGTTTTTTCTTGTTAAATTGTCCACCGACAAAGG 
               
               
                   
               
               
                 CTGGTGGCCTGAAGTCTCCGCAGAGTCTGCCATAAAGTTTGAGCAATTGT 
               
               
                   
               
               
                 AGATGTTGTAGGTTTTTTTTGCAAGTGTTATCGGCGTCCGAAGTTGAAGT 
               
               
                   
               
               
                 GTGACGGGCGCGCAGGAAGGTCAGAAAGCAGCAAGGAAACGGCCAAAGGT 
               
               
                   
               
               
                 ACCAGATAGAAGAAACGGTCTGTTGGGGCTGATTTTGTAGAAACTGATGT 
               
               
                   
               
               
                 TTAATTCACATTTTCTTCACCCGTGGGGGTTCGTTGGGAACCGTCACGAG 
               
               
                   
               
               
                 GCACGTTTGTTGTGGGGCACGTGTGGTTGCAAAATGAGATAAGCAAGGTA 
               
               
                   
               
               
                 GTGTGGTTTGACAGCTTCATATAGGAAGGTGCAAAAAAGTGCAAAGAGAG 
               
               
                   
               
               
                 AAGAATGTAAATTGAAATTGTAATATTCCAATGAGTGAAGTGCTAATTTT 
               
               
                   
               
               
                 GGAAATCTGAGCTTTTTAATGTCTACTCAACTTTGATGTTTCAGTGGATG 
               
               
                   
               
               
                 AAGCCTGTTTGGCGTAAAGTCCACAGGTTTTCGGAGTTTTGGCGAGCATC 
               
               
                   
               
               
                 GACACATAACAAGGCAACAATGCAAAGTCACGAAAATCTCGAACAATGGC 
               
               
                   
               
               
                 GAGTCGTAAATTGGGTCTCTGATTTTCCTAGCGTGAATTGAACAGAAACA 
               
               
                   
               
               
                 GTCCAAGTCCATGCTTGCATTCAGTCACTTGTTTTGAGATGTGGCCGGTG 
               
               
                   
               
               
                 AGAGCCACTGAAAGCGAACCACATACATGTATCCATAATGTACACAATAA 
               
               
                   
               
               
                 AGGCTCCTAGATCAAAGGATCAAGCTCATAATACAAGCAACAACGCCATC 
               
               
                   
               
               
                 GTGTCAGCCGAGCTATTTGAGTGTCACCTGAAGAATAATACCCATACTTG 
               
               
                   
               
               
                 CGCTCTTAAGTGGTAGTATCTGCTGAGCGTCTATCTGATCTGTTGAACTG 
               
               
                   
               
               
                 CTACCACGAGCTTTGGGGATTTTTCGCAGCAATTATACCTGGGCAAATAC 
               
               
                   
               
               
                 AAAGCACAATATTCACAAGCACAGCACTTGTAGGCGACCTCATGCCACTG 
               
               
                   
               
               
                 GTCTGATTAACAAACAATAAGCTTTTGTTTGATAAAACTAATAACGGATC 
               
               
                   
               
               
                 TTGCGAGGGTGATGTAGCTATGAAGAACATGCATGCAACCTCAGCCAGCA 
               
               
                   
               
               
                 TGAGGTATTAGCTCGTAGAATGGCTACGAAGAGACCTAAACAAAAGTAGA 
               
               
                   
               
               
                 AGTAAGTTTAAAGCCTTTCCGATGGAATTTAACAAACTACAAAGTGGAGC 
               
               
                   
               
               
                 AATTCTTTTTGTCGGCTGTTATCTGCTTGAGTGGTATTCATCACGCTGTC 
               
               
                   
               
               
                 TGTGCTCCCGAGGACTCTCCGATACCACAAACTATAATGTTAAATCCACA 
               
               
                   
               
               
                 TATTTTACATGAGCCGAGATGAAGTTATGCAGACTCAACACAAAGGAAAT 
               
               
                   
               
               
                 CAGGGGTCGTATCTTAAGTTCTTCTTGTTTCTAATAATCCTCTCCAGAGG 
               
               
                   
               
               
                 ACTCCATTTGCAGCCACAAATACCTCATCGCGAAAAAGTGCAGCTTCATC 
               
               
                   
               
               
                 TCACTCCATAACTATGTCAGCTGCCGTCGAAAC 
               
               
                   
               
               
                 (SEQ ID NO: 9) 
               
               
                 ATACTTCAACGAAGGAGATTACTTCCAATTTCGAAGCAAAAGATTGGGGT 
               
               
                   
               
               
                 CAGAAGAGCCCAAGTCACCAAGGTCAGGTTATGACTCGACAAAGCAAGCA 
               
               
                   
               
               
                 ACAGCCGACGAAGATAAATCGGAGGGAGATGAGGACGATATATTAAATGA 
               
               
                   
               
               
                 GGGAAACCTCCATTTCCTTGACTTTATTGAGAATAAAACCTTTGGTGTCA 
               
               
                   
               
               
                 ACCCCAGATCAAAGTCGGTCTTCGACCGCCTGGCATACGACTCTGTTGCT 
               
               
                   
               
               
                 TTCATGTCGAACGATGCTGAGGAAGAGGAAAAGGAGAAATCTCTCACGAC 
               
               
                   
               
               
                 TACCTTAGAAGTTCTTGTTGCTCCAGATTCTCCGCCTCCAAGCGATTACG 
               
               
                   
               
               
                 TGATCGATCTTATTCACGAGATATCATCAATTTGCACGGACGTAAAACTC 
               
               
                   
               
               
                 ATGTTACGTTCGCTCAATGTGAAACAAATGTCGAAAGCGTTGAAGCAGAC 
               
               
                   
               
               
                 TGAGGAGGACTACCACAAGCTTGAAAGTTTGGCTCGTCAAGAACGTGAAA 
               
               
                   
               
               
                 CCGACGAAGACAACAATCTGCAAATCACTATGAAACAGACGTCACCTACT 
               
               
                   
               
               
                 AGGCCATCGGTGACGACGCTAAAGACGGGCAGTGGATCTGTGGCTTCTGT 
               
               
                   
               
               
                 TCCGTTCAGGCGTCTGCAAACACTGGAAATCAGTGAGCAGATTCCGCCAC 
               
               
                   
               
               
                 CGTCGCAATTGCAAGGCATGAGATCGTCAACATCGCTCAACACCACAACA 
               
               
                   
               
               
                 TCGGCTCTCAAATTCACGCCTCTTAAGTCTGCAACAACAGGTGGTAAAAC 
               
               
                   
               
               
                 ATTTTCTAAGGGCCTGCTCGAAGACAACAAAGATTTGGACCGGCGCATTG 
               
               
                   
               
               
                 CACAACTTGTGAAAGAAGATGAGAAGAAGAAGCTGAAGGCTGCCAAAGAA 
               
               
                   
               
               
                 GAGAAGCAAAGATTGGCGAAAGAAGAGAAGCTAGCTGCTAAACAAAGCAA 
               
               
                   
               
               
                 GCAGAGGGAAAAGGAAAAACAAAGAGAGGAGTTGTCGCATACGAAGCACA 
               
               
                   
               
               
                 AGGCTACTCCTCTTGAAAGGAACCATACTGAGCCACAAGACTTCTTCTCT 
               
               
                   
               
               
                 ACGAAGCTGAACCGCGAAGATACCGATGAGTCTTCGTTGTTTTCGAAGCC 
               
               
                   
               
               
                 ATCGATCACTTCGAAAGACAAGAAAGGAAGCATTATACTGCGGATCGGAC 
               
               
                   
               
               
                 ACAAACTCAAACATACCGAGCCGCTCAAGCACACGGAGTCTGTTGACAGT 
               
               
                   
               
               
                 GATGTGAGGAGTATTTCCACAACTAAATCGTCCAGTAGCCAAACGTCAAA 
               
               
                   
               
               
                 CACTAGCAAGAAGTCGTCACGCAAGGTTGGATTATTTGGGTTGCGCAAGA 
               
               
                   
               
               
                 GGAATTGAGAAACAAAAGGCAAGAGAGAAAAAAAAAAAAAAAATATATAT 
               
               
                   
               
               
                 ATATATACTAAGTTGGAGGAGAAATCAATCTGCTCCTTGTCAGTGTCTTT 
               
               
                   
               
               
                 TGGAATGATTGCATCGTAATATTCTTTTCTGGAGATTTTTTTTGGGTTTT 
               
               
                   
               
               
                 TTGGAACAGCTGCAACACCATCAACACTCTGAGAAACAACTCCCCGAGAC 
               
               
                   
               
               
                 GCGCCAGCTGCGGCTGCCGCGTAGGAAAGTTGAGCCCCACTGGACTTTTG 
               
               
                   
               
               
                 AGAAGTCTGGGTGCCGGAGGAGGGACCTTCGGCGCGATTTGGGGAGGAAG 
               
               
                   
               
               
                 CAACCTTAGTTGCTTTCTCCAGGATCATTGGATCGCTGAGCAGGGAGTTT 
               
               
                   
               
               
                 CTGTTCTCCTGACGCGTACCGCCTTCTACGGATGCGACGCTAGAAGATAC 
               
               
                   
               
               
                 AGAAACACCTTTTCCAGGACGATCAAACTCGATAGGTGGTTCAGGAGGTC 
               
               
                   
               
               
                 GGGAATCTCGCTCCACGGTACCGGGAGGTCGATTCGCGCCTTCTGCTTGA 
               
               
                   
               
               
                 TGCCGAACGATATTATTCAGCATTTTTCAAAAAGGATCTAAGGGTGTAAG 
               
               
                   
               
               
                 CTTGAGCTTGTAAATGTATTCTAGAACACCGAAGTATTTAAAACTTTGCT 
               
               
                   
               
               
                 TTGAGTCTTTTATTGTATTATTAACTACTGTTTACTATTGCTGTCATATT 
               
               
                   
               
               
                 GAACACCTTTTTGAAATTCGCCAC 
               
               
                   
               
               
                 (SEQ ID NO: 10) 
               
               
                 CTTCGTTGCGTTGAGAAGCGCCTTGTCCAACTTTTTGGACTGCTCTAAAA 
               
               
                   
               
               
                 AGCTGCGAAGCTTCTTAGACTTGGAGTAAACTTGTAGGAATGACACGGCT 
               
               
                   
               
               
                 ACACCTTGAAGAAGGGTGATGGGGGGAGTGACTCCTCCGTGAACGTAGAG 
               
               
                   
               
               
                 CATGTACATGAAAGTTGCGTAGAGGCCACCTTTCGAGCCGGCAACAAAGA 
               
               
                   
               
               
                 TCTCAAATACTTGTGTGTAAATGGGCTTGTTCTCCCACACACGTTCTGAA 
               
               
                   
               
               
                 ACATCTTCGTCGTCGGGATCCTCGTCCTCATCGCTGATTTCCTCATCATT 
               
               
                   
               
               
                 CGATACCTCGTTGCCACCCGGCGACAGCAATCCCTGATTATTGGGGATGG 
               
               
                   
               
               
                 TGTAGAACATGAGCTCATAAACGTTGAGTACGAGTTTACCGAAGTATGCT 
               
               
                   
               
               
                 ATTGCTTCCATCCCGAGCACTCCGTAGTGGATACCAAACAAGAGACTACC 
               
               
                   
               
               
                 AACAGAACGTGTGCCCTGGAAGACGTCGTAAGCCAAAAACTTCGCTAGGA 
               
               
                   
               
               
                 GGAGGTCGGCGACAGTAAATACCTCGAGAAGAATCAGAACCCTATACGTG 
               
               
                   
               
               
                 GCGAAGTTGTAGAAGACCTGGCGAGAGGTTGATATCTGATGCGGAATCAT 
               
               
                   
               
               
                 GTTCGTGATTTTCATCTGAAACGAGTCAAGACGGTCCGTCATGATGATAT 
               
               
                   
               
               
                 GAAACACTTTGAACATGATCGTGAGGTTGATCCACACTATATTGAGAAGA 
               
               
                   
               
               
                 AGATTATCGTCACTAAACATGATTATGAGAAGATTAATAAGGTAGAAGGG 
               
               
                   
               
               
                 TAACTCCTCCGAAATATGCTCAATTTCAATGATCCGAAGCTCTCTGAAGA 
               
               
                   
               
               
                 TCAATTTGATCCCTGCAAACCCCATGAGAATAAAGCAACTTACCACGAAG 
               
               
                   
               
               
                 TTGAGGAGAATTAGGAGTCTCACGCTCTCCGTGAGCTCGTACATCATGGT 
               
               
                   
               
               
                 AAGATAATCGACAGAGTTCATGGCCAGAACCACGAGCGATGCAGCGAAAA 
               
               
                   
               
               
                 GGCCGAAGCTAATGCCGCCATACACTGCAATTGCAATGGTGCCCTTTCGA 
               
               
                   
               
               
                 GGCATGGTTATGGTGTTTTGTCTTTCTTTTGTGGGGACGAAAGGTGTGGA 
               
               
                   
               
               
                 AGCCGGAAGTAAACACCTCACCTTCGCGATAATCCTAGATCTACGCTAAC 
               
               
                   
               
               
                 TTGCGCATAGAGGGTCGTGCGCGCAAAATTTTCCCCAGACTTCCAATTAG 
               
               
                   
               
               
                 TGGTCGCTTGAGTCTATTAAAGTTGCGGAATTCGGGACTTATGGAGGTCA 
               
               
                   
               
               
                 TTGCGTAATGGTGACATTTCAGTTCGTCTTCTTTTGATGTCTGGAATCAT 
               
               
                   
               
               
                 GCTCTGTCCGTTTAATTGCCTCATAGGCAGCCTCAGTGGCAGTCTCATGG 
               
               
                   
               
               
                 GCAGTCGCAGAGGTGATCTGGATGCTGGTGAAGTTGCCGGGCGCTTCAAT 
               
               
                   
               
               
                 TGAGCAGGTGATCTTTGTAGCTCTGACAATGATTGGGAAAATATTTTGAG 
               
               
                   
               
               
                 GTTCTTCTCGGTGTGATTTAAGTCGCGAGAAATTCCGTGGTAGTTCTTAT 
               
               
                   
               
               
                 CGATCATGTCGGATACAATATGTGCCTTTTGAGTTGCAGCTTTCTCTATA 
               
               
                   
               
               
                 GAGTCCGCCTGGCTGCGACTAGTATTGGTAATATCAGCCAAAGCATTATC 
               
               
                   
               
               
                 AATAACTTGTTTGGTCTCCTTCTGTAATCTTCCAAAGGCTCCCTGCACAT 
               
               
                   
               
               
                 CTTTGGTGGCAACCTCTTTGAAGGACTTGAGATCCGTGGCCACTGACTTG 
               
               
                   
               
               
                 GTCTTTGTTGATATCGACTGTAGCGTTTGGGAATTTTGATGAATGTAGGC 
               
               
                   
               
               
                 CTGATACTCTTTGCTTAGAGTTGTCTTCTCTTCTTGCATGAATTCTGTTG 
               
               
                   
               
               
                 CACTTTCCCTAACCGCCTCGGCATTGGCGTCCTTGCTTGACTTCACGAAC 
               
               
                   
               
               
                 CCGTCAAATGCTAGGTGCATTTGTTGCATATATTGCTCCTTAAGTGCCTC 
               
               
                   
               
               
                 ATACGTCTCTGCCATACGAGCCTTGAACTTCTTCTCAAAGGCCTTGTACA 
               
               
                   
               
               
                 TTGCGGAGTCTTCCAGAAGATGTGTTGAGTTGATGTAATCCGTTAGTTGC 
               
               
                   
               
               
                 TCATTCCTCTTGTTCACTCCAGCTACGTGCAGCGAGAAATCGAGGTTTTG 
               
               
                   
               
               
                 ACAAACATTATGAAGGGTAGAAAAGTCCATCAGATTAGCGAGAACCTGCT 
               
               
                   
               
               
                 CGAAGGCTGATGTCTCCTTCTCTACTTTGGAAGAAACATCATTTAATTTG 
               
               
                   
               
               
                 CCCTCGAGAGTCTTCTTAAAAAGGCAAAGATGCGTTTGAACGACGGTGGG 
               
               
                   
               
               
                 GATCTCCTTCACATTCAGCAGCACCTGAGTTAGGTCATTAAGATTGTTAA 
               
               
                   
               
               
                 TCACCTCATTAACGTCCCTAATGATACTCAGAATTCCAGTTCCTGTAACA 
               
               
                   
               
               
                 TTAGAGACTCGCTCTTTTTGAGAGCTGTATTTGTCTTTTAAATGCATGAA 
               
               
                   
               
               
                 CTCTTTTGTAGTCTTGTGAAGCTCAGCGTCTTTAGCTGCGAGGTCCAGCC 
               
               
                   
               
               
                 TGCTTTTCGAGTTCTCGCTAAGTGTTTGCTCGATCTGTAGTTTGAGTTCT 
               
               
                   
               
               
                 GCAATATCCTTGTCCCTCCCTTGAAGTTTGGCGTGTAGCCCTGTAATCTG 
               
               
                   
               
               
                 AGTGTCTTTCTCCTTCAACTCGGTTCTAAGTTCCGCAATTTTCCTTTCAA 
               
               
                   
               
               
                 AGTCCTCGTAGTTCTGAAGACTGATCCTGATGCTGTTGTCTTTGCCTTTT 
               
               
                   
               
               
                 GTAGCCAATAAGTCTCTATTCAATCTAGAAATCTCCGCTGATAACTCCCT 
               
               
                   
               
               
                 TATCTTTGTACGTTTGAGAACGAGCTCGCTGTCGGCCGTCGATTGTGGTA 
               
               
                   
               
               
                 TATTTTTGATATTCTTCGCCTTGGAAGCGTAAGTTAGCGTCAGCATCGTT 
               
               
                   
               
               
                 TCCATTAAGTTCAGCTTTGCCGGGGAAATGGTAGCAATCAATGCGGTTTT 
               
               
                   
               
               
                 GGTCCGCCCTCCAATAGAGCCTTGAAGAAGACGGGTGAGTTTAGACTCTC 
               
               
                   
               
               
                 TATAAGGTATATGTCTAGGTTCTTTGCCTTCACTCAATGCACTGATGACC 
               
               
                   
               
               
                 TTTCCCAAAGTCAAAAGACTCTGGTTGATCAGGCCCGCTTCTTTGGCACT 
               
               
                   
               
               
                 AGCATCGGTAGCGCCAGACTTGATGATATCTTCCAGCCCCGCGAGATCCA 
               
               
                   
               
               
                 CCAAATTCATTTTTGACAGCCGCACCACCTCTTGCCCTGACGACGACATT 
               
               
                   
               
               
                 ACTGTTTTGTGAAGCGTTATGGTGAAAATGGTGTGAGAACGTGATGAGCG 
               
               
                   
               
               
                 GGAGTTGAGTTTAGTGGTACCCATCTTCCTCTTCCCTAGGCACTTTTGTA 
               
               
                   
               
               
                 GCATCTCAAACCCCAGCTTCGCATCCACCACGTCAAGCTCATACAAATTC 
               
               
                   
               
               
                 TGGATCATTGTTCCCCTTCCATCTCTCGAACCATCCCCTAGGAGCCTTAG 
               
               
                   
               
               
                 TTTCGGCTTTTTTGAGTTCAACTCAAGCTCATCGTTGACGAGATCATGGA 
               
               
                   
               
               
                 GCTCTTCCTTGTACAATTCCACACACGACAACTTGACACAGATGTCGTCT 
               
               
                   
               
               
                 TTGGCCACCTGAAAAAGCTCTTGCAACACACGAGGCACAATACCCGCATG 
               
               
                   
               
               
                 CTCTCCCACTAAATCCCCCAACATTGTATACGTCTTTCCTGAGCCTGTCA 
               
               
                   
               
               
                 GGCCATACGCAAGGATAGTGACATTCATGCCTGCCATGAAATCTCGAAGT 
               
               
                   
               
               
                 AACGGACGAGCAATGTTTTTGTAAATGAGTTCCTGATCAGCATTGGCACC 
               
               
                   
               
               
                 ATAGACTTGGTCTAGTGTAAATACTTTGCCTGAACCGTCTGATCCAGAGC 
               
               
                   
               
               
                 CAAAGGAAGTGTTGGGGGAAGCATTCACGCTAACGTAGGGTTCATCCGTG 
               
               
                   
               
               
                 GAACAGAAATCGTCAGGCACCGAGACCACAATCGGGGACTGGGCGGCTAT 
               
               
                   
               
               
                 CTCCAGCTCAGTTCTTCTTCGCACACGGGCGCTGACCTGGATTTTGTCGG 
               
               
                   
               
               
                 ACATATAGTACAGTTTTTTTTTTGTAGTCTTTCTTTGTGGAGATTATGGT 
               
               
                   
               
               
                 GATGTTTATGTTTGTTTACGATGGGCGGCCTGTGTGCAGGTCGGAACGGT 
               
               
                   
               
               
                 CAAAGCATGTAGAGTGTCTAGAGTCTTTATCGATGAATGGAAGTAGAGGA 
               
               
                   
               
               
                 GGTAAAATTCTAATAGTGAGATTCTTTTTTCGATGGACGTGTTTTTGTTT 
               
               
                   
               
               
                 ACGTTCCTCGTACTTAGTTATGTTTCGGGTTTAATGGTGTTTAGTGAGAA 
               
               
                   
               
               
                 ATGGCTGCAAAATGCAAAATGCTGCGAAAAAGTATAGATCAGAGAAAGAC 
               
               
                   
               
               
                 AATACTACTGTCTGATAAAAAACAAAAGTTGATGATAAGAATACCAGAAT 
               
               
                   
               
               
                 TTGTACTCACATATAGGAAATCACCTAGAGTTTGATATATAATCTGACAC 
               
               
                   
               
               
                 AGCAATGTCAAAATCGCTTTTACCACGTGCTATAAAAAGTTTCTAAGGAC 
               
               
                   
               
               
                 GTCACCTTCTCGATATAAGATAAGCATTCTGTGAACGGCTGTTAGGAAAG 
               
               
                   
               
               
                 AGCATAGTGAGGTATTTCAGTTGAAATACGTATGCCAAAAAAAAGGCAAC 
               
               
                   
               
               
                 CATTAATTAGATTCCACGTGGTTCACTACACTGAAAACAGATGAACTGTC 
               
               
                   
               
               
                 TACAATACAGTCTGTCAGTGCAATGCCTACCAGTTGCTGGTGGCATAGCA 
               
               
                   
               
               
                 CCTCACGAATGGATATATTCGCTACCAGGTTTTGTACAGCTCAAACCACT 
               
               
                   
               
               
                 ACATAGCTGGTTTCTGTCAACCTCGTCGCTATCAACTAACAAACTCTTTT 
               
               
                   
               
               
                 ACTAAAAAAGAGAAGCTCTTCTTTCACTACACGTACTGTACTAGCCTTCG 
               
               
                   
               
               
                 CTCGTGTGACGATTAGCCAGCCGTACTTTTCCCCATCACCGTAAACCATC 
               
               
                   
               
               
                 TATTGATGTTCATATATACAGATTCCAATGACGACGCCAATGGCAATGCC 
               
               
                   
               
               
                 ACGAGCCCCCAAAAACCTCAATCAAAGAAATCATCGTCATCTTCAT 
               
            
           
         
       
     
     Among from the sequences represented by SEQ ID NOs: 7 to 10, the nucleotide sequence represented by SEQ ID NO: 7 was selected as a sequence having a length of about 1 kbp and most suitable for a LAMP reaction on the basis of the experience of the present inventors. As the result of the BLAST search in the National Center of Biotechnology Information of the United States, it was confirmed that the nucleotide sequence represented by SEQ ID NO: 7 was the genome of  Candida auris  ( C. auris ) containing a pyruvate: ferredoxin oxidoreductase domain (accession number of the genome: XM_018317007). 
     The DNA fragment represented by SEQ ID NO: 7 was amplified with EcoRIdAmp (registered tradename) PCR Master Mix (manufactured by Takara Bio Inc.) using  C. auris  JCM15448 T that served as a template and a pair of primers consisting of Auris F (SEQ ID NO: 11: GCTATGCCGCTAGCAACG) and Auris R (SEQ ID NO: 12: CACTACAGCAGGATCAACGG). 
     In order to produce a pTAC-2Auris plasmid that was specific to the DNA of the fungus, an amplicon was purified with QAquick (registered tradename) PCR purification kit (Qiagen, Venlo, The Netherlands) and was then cloned into a pTAC-2 vector using DynaExpress TA PCR cloning kit (BioDynamics Laboratory Inc., Tokyo, Japan). The cloned nucleotide sequence was confirmed using ABI PRISM (registered tradename) 3130xl Genetic Analyzer (Applied Biosystems, Foster City, Calif., USA). 
     A candidate LAMP primer set was designed using a 192-bp fragment (gene locus XM_018317007: located from 774b to 965b) as a target and using PrimerExplorer V5 software (https://primerexplorer.jp/lampv5/index.html). The decided LAMP Auris primer set (also sometimes referred to as “LAMP Auris primer set”, hereinafter) is shown in Table 1. 
     
       
         
           
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Primer 
                 Sequence (5′→3′) 
               
               
                   
               
             
            
               
                 FIP 
                 AGGCTACTGAGCTTGCTGGTGTAACCAAACCAACAGGAGAGG 
               
               
                   
                 (SEQ ID NO: 1) 
               
               
                   
               
               
                 BIP 
                 ACGGTTTCAGGGTTAGCATGGCTCAACAAAGTCGCTGGTACA 
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
               
               
                 F3 
                 GGGAAAGGAACCCTGACCT 
               
               
                   
                 (SEQ ID NO: 3) 
               
               
                   
               
               
                 B3 
                 GGACACAGCATTCGAAGTGT 
               
               
                   
                 (SEQ ID NO: 4) 
               
               
                   
               
               
                 Loop-F 
                 CATCTCGAAGGCCTCGGT 
               
               
                   
                 (SEQ ID NO: 5) 
               
               
                   
               
               
                 Loop-B 
                 CACATACTCGAACGGAGTC 
               
               
                   
                 (SEQ ID NO: 6) 
               
               
                   
               
            
           
         
       
     
     It should be noted that the designing of the LAMP primers is not always easy even when a specialized software is used. For example, in addition to the basic LAMP primer set represented by SEQ ID Nos: 1 to 4 shown in Table 1, a primer set including the polynucleotides represented by SEQ ID NOs: 13 to 16 can be mentioned as another example of the basic LAMP primer set which can be designed on the basis of SEQ ID NO: 7. The primer set was confirmed to cause no  Candida auris  gene amplification reaction by the LAMP method. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 13) 
               
               
                   
                 GTGGTATCTTCGGATGCTGGCTCCAGCGAGGAACAACGAA(FIP) 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 14) 
               
               
                   
                 ACAACCTTGCGTGCAGACGAAGGGTTCCTTTCCCATTTGC(BIP) 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 15) 
               
               
                   
                 AGAAACGGAACCGAAACGG(F3) 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 16) 
               
               
                   
                 CCTCTCCTGTTGGTTTGGTT(B3) 
               
            
           
         
       
     
     &lt;Example 2&gt; Amplification Reaction by the LAMP Method 
     With respect to a cultured  Candida auris  specimen, an amplification reaction by the LAMP method was run using Loopamp Turbidimeter RT-160C (manufactured by Eiken Chemical Co., Ltd.) at 56° C. for 90 minutes. The reaction was terminated by deactivating the DNA polymerase at 80° C. for 5 minutes. Each of the reaction solutions was prepared by mixing 12.5 μL of 2× Reaction Mix (manufactured by Eiken Chemical Co., Ltd.), the primers of LAMP Auris primer set (40 μM of FIP, 40 μM of BIP, 20 μM of Loop-F, 20 μM of Loop-B, 5 μM of F3, 5 μM of B3), 1 μL of BstDNA polymerase, 2 μL of a sample DNA solution and distilled water together, and the total volume of the reaction solution was adjusted to 25 μL. 
     In order to determine the detection limit of the LAMP Auris primer set, pTAC-2Auris was serially diluted (1×10 0  to 10 10  copies/μL) and was used as a template in triplicate reactions. The detection of an amplification product was carried out with Loopamp Turbidimeter RT-160C. 
     The results are shown in  FIG. 1 . 
     As shown in  FIG. 1 , according to the LAMP method using LAMP Auris primer set, it was confirmed that pTAC-2Auris was detected and the sensitivity was high even when the concentration was 2×10 1  copies or less per one reaction. With respect to the extraction of DNA from the fungal cells, it was also confirmed that the DNA was extracted with high efficiency even when the fungal cells were treated with a DNA extraction reagent directly (i.e., without being disrupted). 
     &lt;Example 3&gt; Evaluation of Specificity of LAMP Auris Primer Set 
     In order to evaluate the specificity of the LAMP Auris primer set to  Candida auris  ( C. auris ), 57 strains in total of 38 species (20 kinds of filamentous fungi and 18 kinds of yeast strains) were tested (Table 2). 
     The full-length DNA of a template strain was extracted and purified in accordance with the method disclosed in Non-Patent Literature 4. With respect to the yeast strains, an aliquot of a colony proliferated on Sabouraud dextrose agar (SDA) was suspended in 25 μL of distilled water, and the resultant suspension was heated at 100° C. for 15 minutes and was then centrifuged for a short time. In each of the reactions using the LAMP Auris primer set, 2 μL of a supernatant or purified DNA was used as a template. 
     The results are shown in Table 2. 
     
       
         
           
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                 LAMP 
               
               
                   
                   
                 Auris 
               
               
                   
                   
                 reaction 
               
               
                 Species 
                 Strain No. 
                 resuit 
               
               
                   
               
             
            
               
                 
                   Acremonium curvulum 
                 
                 NBRC32242 
                 − 
               
               
                 
                   Aspergillus fumigatus 
                 
                 TIMM0108 
                 − 
               
               
                 
                   Aspergillus niger 
                 
                 TIMM0115 
                 − 
               
               
                 
                   Candida albicans 
                 
                 LSEM11-828 
                 − 
               
               
                 
                   Candida auris 
                 
                 CBS12323, CBS12372, 
                 + 
               
               
                   
                 CBS12766, CBS12767, 
               
               
                   
                 CBS12768, CBS12769, 
               
               
                   
                 CBS12770, CBS12771, 
               
               
                   
                 CBS12772, CBS12773, 
               
               
                   
                 CBS12774, CBS12775, 
               
               
                   
                 JCM12373, JCM15448 ? , 
               
               
                   
                 LSEM52-3435, LSEM52-3449 
               
               
                   Candida duobushaemulonii  * 
                 CBS7799 
                 − 
               
               
                   Candida famata  * 
                 NBRC0083, NBRC0623 
                 − 
               
               
                 
                   Candida glabrata 
                 
                 CBS138, NBRC0005 
                 − 
               
               
                 
                   Candida guilliermondii 
                 
                 TIMM0257 
                 − 
               
               
                   Candida haemulonii  * 
                 JCM3762 
                 − 
               
               
                 
                   Candida krusei 
                 
                 TIMM3378 
                 − 
               
               
                   Candida lusitaniae  * 
                 NBRC1019, NBRC10059 
                 − 
               
               
                 
                   Candida parapsilosis 
                 
                 ATCC22019 
                 − 
               
               
                 
                   Candida pseudohaemulonii 
                 
                 JCM12453 
                 − 
               
               
                   Candida sake  * 
                 NBRC0435 
                 − 
               
               
                 
                   Candida tropicalis 
                 
                 ATCC750, TIMM0313 
                 − 
               
               
                 
                   Chaetomium globosum 
                 
                 TSY-0369 
                 − 
               
               
                 
                   Cladosporium carrionii 
                 
                 TIMM3048 
                 − 
               
               
                 
                   Cunninghamella bertholletiae 
                 
                 TIMM3392 
                 − 
               
               
                 
                   Exophiala ieanselmei 
                 
                 TSY-0396 
                 − 
               
               
                 
                   Fusarium oxysporum 
                 
                 TSY-0351 
                 − 
               
               
                 
                   Fusarium solani 
                 
                 TSY-0403 
                 − 
               
               
                 
                   Malassezia furfur 
                 
                 CBS1878 T   
                 − 
               
               
                 
                   Malassezia restricta 
                 
                 CBS7877 
                 − 
               
               
                 
                   Microsporum gypseum 
                 
                 NBRC5948 
                 − 
               
               
                 
                   Mucor circinelloides 
                 
                 TIMM3177 
                 − 
               
               
                 
                   Paecilomyces variotii 
                 
                 NBRC4855 
                 − 
               
               
                 
                   Pseudallescheria boydii 
                 
                 TIMM0886 
                 − 
               
               
                   Rhodotorula glutinis  * 
                 LSEM 20-1447 
                 − 
               
               
                 
                   Rhodotorula minuta 
                 
                 TIMM6222 
                 − 
               
               
                 
                   Saccharomyces cervisiae 
                 
                 LSEM14-1013 
                 − 
               
               
                 
                   Scopulariopsis brevicaulis 
                 
                 NBRC4843 
                 − 
               
               
                 
                   Scopulariopsis brumptii 
                 
                 NBRC6441 
                 − 
               
               
                 
                   Scytalidium lignicola 
                 
                 NBRC104988 
                 − 
               
               
                 
                   Trichophyton benhamiae 
                 
                 SM103 
                 − 
               
               
                 
                   Trichophyton mentagrophytes 
                 
                 TIMM2789 
                 − 
               
               
                 
                   Trichophyton rubrum 
                 
                 TIMM2659 
                 − 
               
               
                 
                   Trichophyton tonsurans 
                 
                 NBRC5928 
                 − 
               
               
                   
               
               
                 +: positive, 
               
               
                 −: negative, 
               
               
                 * a species which  Candida auris (C. auris)  has been misidentified as in common tests. 
               
            
           
         
       
     
     By the LAMP method using the LAMP Auris primer set, the occurrence of amplification was confirmed with respect to all of 16 kinds of  C. auris  strains. In contrast, with respect to all of the remaining filamentous fungi and yeast strains including fungi which  C. auris  has been commonly misidentified as (i.e., fungi that have not been identified as  Candida auris  ( C. auris ) in the conventional test methods: marked with asterisks in Table 2), the occurrence of amplification was not confirmed. 
     In order to verify the quality of the DNA templates used in the reaction with the LAMP Auris primer set, the DNA templates were separately subjected to the LAMP reaction using panfungal LAMP primer set (Non-Patent Literature 4). As a result, the occurrence of amplification was detected with respect to the templates from all of the test species. 
     &lt;Example 4&gt; Detection of  Candida auris  ( C. auris ) in Clinical Specimen by LAMP Method 
     The LAMP method using the LAMP Auris primer set was applied to a clinical specimen. The test was carried out on an ear swab specimen obtained from otitis induced by  C. auris  LC318417 (Non-Patent Literature 5). 
     An ear swab specimen was placed on the surface of SDA agar and was then cultured at 37° C. A small cream-like colony (cells) thus obtained was identified as  Candida auris  ( C. auris ) by the LAMP method of the present invention, MALDI-TOF MS (Bruker Daltonics K. K., Kanagawa, Japan) and rDNA sequencing. 
     At the same time, it was attempted to detect  Candida auris  ( C. auris ) DNA directly from a clinical specimen without carrying out culturing in the following manner. A cotton swab was placed in a 2-mL-microtube, was then washed with 1 mL of physiological saline supplemented with 0.05% Tween 80, and was then shaken for 10 minutes. A suspension thus obtained was centrifuged at 20,000 g for 10 minutes to produce pellets, the pellets were washed with 100 μL of physiological saline, and the full-length DNA was extracted using Kaneka Easy DNA Extraction kit version 2 (manufactured by Kaneka Corporation) in accordance with the instructions by the supplier. A nucleic acid amplification reaction by the LAMP method was carried out using the extracted DNA (2 μl) as a sample nucleic acid for an LAMP reaction solution and using the LAMP Auris primer set. The reaction conditions and the method for the detection of an amplification product were the same as those employed in Example 2. 
     The results are shown in  FIG. 2 . 
     As shown in  FIG. 2 , the amplification product produced by the LAMP method from the clinical specimen was detected in 27 minutes. It was confirmed that the LAMP method using the LAMP Auris primer set was applicable to a clinical specimen directly. The whole identification process including the direct extraction of DNA (i.e., extraction without disruption) was completed within about 1 hour, and it was confirmed that rapid detection could be achieved. 
     &lt;Example 5&gt; Detection of  Candida auris  ( C. auris ) in Environmental Sample by LAMP Method 
     The applicability of the LAMP method using the LAMP primer set to an environmental research was also evaluated. About 25 μL of physiological saline containing 1×10 6  cells of  Candida auris  ( C. auris ) was dried in a clean petri dish, and the cells were collected by a wiping method that had been optimized for environmental sampling use (samples A to D). 
     The cell amounts and the collection rates of samples A to D are shown in Table 3. 
     
       
         
           
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Sample 
                 Amount of collected cells (cells/μL) 
                 Collection rate (%) 
               
               
                   
               
             
            
               
                 A 
                   5 × 10 2   
                 51 
               
               
                 B 
                  5.6 × 10 2   
                 57 
               
               
                 C 
                 6.25 × 10 2   
                 63 
               
               
                 D 
                 3.75 × 10 2   
                 38 
               
               
                   
               
            
           
         
       
     
     The handling of swabs and the extraction of DNA, excluding the shaking, were carried out in the same manner as in Examples 2 and 4. With respect to samples A and B, a vibration shaker was not use. With respect to samples C and D, the vibration shaker was used for 10 minutes. The reaction conditions for the nucleic acid amplification reaction by the LAMP method and the detection of an amplification product were the same as those employed in Examples 2 and 4. 
     The LAMP reaction times and the detection results for the samples are shown in Table 4 and  FIG. 3 . 
     
       
         
           
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                   
                   
                 Time required until LAMP 
               
               
                 Specimen 
                   
                 auris-positive reaction occurs 
               
               
                 No. 
                 Specimen 
                 (min) 
               
               
                   
               
             
            
               
                 1 
                 Distilled water 
                 Positive reaction 
               
               
                   
                 (negative control) 
                 does not occur 
               
               
                 2 
                 Sample A 
                 21 
               
               
                 3 
                 Sample B 
                 16 
               
               
                 4 
                 Sample C 
                 22 
               
               
                 5 
                 Sample D 
                 23 
               
               
                 6 
                   Candida auris  DNA 
                 20 
               
               
                   
                 (positive control) 
               
               
                   
               
            
           
         
       
     
     As shown in Table 4 and  FIG. 3 , with respect to each of the samples (A to D, positive control), an amplification product was detected (LAMP-positive) 20 minutes after the start of the nucleic acid amplification reaction by the LAMP method, and it was confirmed that  Candida auris  ( C. auris ) DNA was detected. It was suggested that the LAMP method using the LAMP Auris primer set was applicable to environmental researches and enabled rapid and accurate detection. 
     From the results of Examples mentioned above, it was confirmed that, according to the LAMP method using the LAMP Auris primer set, all of  C. auris  strains were able to be identified with 100% of specificity and were able to be detected distinctively from closely related species thereof with high reliability. Furthermore, the target DNA was detected at a concentration of 2×10 1  copies per reaction, and therefore there was no technical problem about the use of an amplification device and the results were obtained within a short time. Furthermore, it was also confirmed that the detection from a clinical specimen was possible and therefore the time necessary for culturing or extraction of DNA was shortened, and early diagnosis became possible. In recent years, a portable LAMP amplification device and the like are commercially available. Therefore, according to the LAMP method using the LAMP Auris primer set of the present invention, the rapid and accurate detection of  Candida auris  ( C. auris ) becomes possible in, for example, environmental researches in medical facilities where the environmental control is critical.