KR19980701336A - NUCLEIC ACID PROBES FOR THE DETECTION AND IDENTIFICATION OF FUNGI - Google Patents

Abstract

Nucleic acid labels and primers are described to detect food and beverage damage, as well as fungi causing disease in humans and animals. The labels can detect rDNA or polymerase chain reaction products from a number of fungi present in clinical, environmental or food samples. Acremonium species, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergillus ustus, Beauvaria, Blasto Candida dascalans , Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Chrysosporium spp., Cladosporium spp., Coccidioides immitis, Cryptococcus neoformans var gattii serotype B, Crytococcus neoformans serotype A, Cryptocous terre , Fusarium species, Filobasidium capsuligenum, Filobasidiella (Cryptoccus) neoformans var bacillisopora serotype C, Filobasidiella (Cryptoccus) neoformans var neoformans serotype D, Filobasidium uniguttulatum, Geotrichum species, Histoplasma capsulatum, Malbranche species, Penicillium species Pseudallescheria boydii, Rhizopus species, S Nucleic acid hybridization markers specific for porothrix schenkii, Scopulariopsis brevicaulis, Scopulariopsis brumpti, Saccharomyces cerevisiae and Trichosporon beigelii are also described.

Description

Nucleic Acid Labels for Detection and Identification of Fungi

[Technical Field]

The present invention is directed to the design and composition of two nucleic acid probes capable of detecting many different fungal organisms present in clinical, food, environmental and other samples. It is about. In addition, the present invention is Acremonium species, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguistus, Aspergillus unguistus , Bipolaris species, Blastoschizomyces species, Blastomyces dermatitidis, Candida albicans, Candida glabrata, Candida guilliermondii, candida kefyr, Candida kursei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Chrysosporium species, Cladosporium species, Coccidioformypto var. serotype) B, Cryptococcus neoformans serotype A, Cryptococcus laurentii, Cryptococcus ferreus, Curvularia species, Fusarium species, Filobasidium capsuligenum, Filobasidiella (Cryptococcus) neoformans var bacillispora serotype C, Filobasidiella (Cryptans varigformos neoformans Dupocococulus) Geotrichum species, Histoplasma capsulatum, Malbranch ea, Mucor, Paecilomyces, Penicillium, Pseudallescheria boydii, Rhizopus, Sporothrix schenkii, Scopulariopsis brevicaulis, Scopulariopsis brumpti, Saccharomyces cerevisiae and Trichosporon beigelii. .

Fungi are strongly distributed eukaryotic microorganisms. Fungi play an important role in degrading plant material in nature, while they also cause damage to foods, beverages and medicinal preparations. Of the 100,000 strains published by mycologists, about 150 are pathogens for humans and animals. The increase in the incidence of AIDS (AIDS) and the development of new therapies for hematologic malignancies and organ transplants have increased the number of patients with immunocompromised. The patients are at risk of developing a bacterial infection, which can cause death within a few days if not diagnosed or treated promptly. The number of antifungal drugs is limited and the toxic side effects of the antibacterial agents on the patient are greater than the toxic side effects of comparable antibacterial therapies. For such patients, an early diagnosis and treatment of fungal infections is urgent. Together with Sarosi and Davies, books written by Kwon-Chung and Bennett provide insights into the medical importance of fungi.

Fungal organisms are identified by morphology (morpho1ogy) and nutritional properties. Fungi can grow anywhere from two days to several weeks in culture, and often the organism can have a radically different form depending on its growth conditions. Such mutations make it difficult to timely identify even traditionally trained specialists, and the rapid identification of genus and species hinders the treatment of patients with medical benefits.

The incidence and distribution of major pathogen fungi vary by geographic location. Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidiodes immitis, Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Pseudallescherisa boydii and Sporothrix schenkii have several causes of infection of mycotic.

Aspergillus fumigatus is one of the top three causes of systemic fungal infections treated in hospitals. The fungus usually affects patients with intestinal transplantation, severe leukemias and burns, and can be very fatal if not diagnosed quickly. Over 150 species of Aspergillus are present in soil, air and water, and accurate detection of Aspergillus fumigatus has become very important. Aspergillus clavatus, aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus nuguis and Aspergillus ustus are responsible for most of the diagnosis of Aspergillus species present in clinical specimens. You can. Aspergillus flavus, Aspergillus fumigatus and Aspergillus niger have been associated with human disease along with Aspergillus fumigatus, the dominant pathogen in North America. There are several immune tests for Aspergillus fumigatus, but these tests have limited sensitivity and specificity. There is also a report on the development of Polymerase Chain Reaction (PCR) based on testing of Aspergillus fumigatus based on amplification of the Asp fl antigen gene and the ribosome intergenic spacer (Spreadbury et al.). . The Spreadbury technique is based on PCR amplification of 401 base pair (bp) fragments that span large subunit rRNA / intergenic spacer regions. The technique relies on a pair of primers that specifically amplify DNA only from Aspergillus fumigatus and are not useful for identifying other fungi.

Aspergillus dermatitidis is present in soil, typically bird droppings and animal droppings. Infections often occur at the construction site, and subsequent lung infiltration and pneumonia are generally fatal for immunocompromised patients. Diagnosis by culturing may take several weeks, and the organism may be mistaken for other fungi as the case may be. Existing immunodiagnostic tests are incredible and require fast and reliable DNA based diagnostic tests. Similarly, Histoplasma capsulatum is present in soil and at least 20% of the North American population is known to be infected. Most infections begin in the lungs and spontaneously recover, but can occasionally spread to other organs. AIDS patients show an increase in the number of cases of histoplasmosis. Immune tests are often difficult to diagnose because they are negative during the first four to six weeks of infection. Coccidioides immitis is found in abundance in the soil of the southwestern United States. Dust, storms, farms, building constructions, earthquakes and even walking trips have been linked to disease outbreaks. Infection of the lung following cavitation and coccidioidomycosis of metastasized lip bales appear. Meningitis is usually fatal, and mortality is greatest in debilitated hosts when combined with other fungi. In humans, the four sera of Cryptococcus neoformans are A, Cryptococcus neoformans var gatti serotype B, Filobasidiella (Cryptococcus) neoformans var bacillispora serotype C and Filobasidiella (Cryptococcus) neoformans var meoformans serotype D. The incidence of the disease is increasing rapidly, accounting for more than 10% of patients with HIV infection that cause cryptococcosis. There is a need for DNA markers capable of detecting all four serotypes for early diagnosis and treatment of survival-threatening infections such as Cryptococcal meningitis. A report by Stockman et al. Publishes commercial test methods for Histoplasma, blastomyces, Coccidioides, and Cryptococcus based on 18S rRNA (Gen-Probe, Inc., San Diego, CA). The authors report sensitivity in the range of 87.8 to 100% and specificity of 100%. One disadvantage of the label is that the label is used for rRNA extracted from fungal culture. Because some fungi need more than three weeks to incubate, the technique cannot be used to expedite diagnosis until cultures become useful.

Candida albicans is one of the most common causes of fungal infections in humans. The fungus is present in the pathways of the respiratory, gastrointestinal and female genitalia of healthy humans and acts as an opportunistic pathogen in debilitated humans on steroids and chemotherapy. Diabetes and the inherent catheter cause other susceptible infections. Immunocompromised hosts show rapid hematogenous transmission of fungi. The number of patients and mortality is very high in the untreated cases. Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis and Candida tropicalis are known to occur in the human body. DNA markers that can identify each species will reduce the need for much blood culture and biochemical differentiation.

Recent advances in molecular biological technology have led to an approach of microbial detection and identification based on the DNA sequence of ribosomal genes. Commonly used detection techniques are direct amplification of ribosomal DNA (rDNA) genes by polymerase chain reaction, or reverse transcryption of ribosomal RNA (rRNA) to complementary DNA (cDNA) following polymerase chain reaction of cDNA. It includes. Ribosomes are a composite of protein species that act in the translation of single rRNA and messenger RNA into proteins. The theory of evolution is consistent with the interpretation that all existing life evolved in one organism. Therefore, all organelles contain rRNAs, which are involved by evolution. The evolutionary process is that each species' organ shows a single region of sequence within its own ribosomal gene. The presence of specific regions of the single species specifically binds under hybridization conditions and devised DNA markers to identify DNA amplified from a single species by the polymerase chain reaction. For the purposes of the present application, the word primer means a nucleotide sequence that can be expanded by polymerization polymerized in a template, and a label is a nucleotide sequence capable of detecting its complementary sequence by hybridization. Means. Also, for the purposes of the present application, the phrase nucleotide sequence will include DNA or RNA types or modifications thereof. Moreover, one skilled in the art will recognize that primer sequences can be used as markers and vice versa. The use of nucleic acid hybridization to detect specific nucleic acid sequences of interest has also been published by Kohne (US Pat. No. 4,851,330,7 / 1989).

In prokaryotes and eukaryotes, ribosomal RNA and corresponding rDNA genes are identified by the size of the RNA. The magnitude is related to the sedimentation velocity and the S value. As a result, these values are 5S, 16S and 23S for prokaryotes and 5S, 5.8S, 18S and 28S for eukaryotes. Since all ribosomes perform the same function essential for cell survival, the sequences of ribosomes are mostly preserved, but some regions of each ribosomal species are not affected by functionalization and require more changes. The hypervariable regions identify different species in the same genus. As mentioned in the above references, there are several reports that intergenic spacers between 5S, 18S and 5.8S and 28S rDNA have been used for identification and detection of fungi (Holmes et. A1., Hopper et al. Hopfer et. Al., Lott et. A1., Maiwald et. Al., Makimura et.al., Mitchell et al., Nakamura et al. (Nakamuraet.al.)). Holmes publishes PCR based on adjacent non-transcribed spacer regions. Co-amplification of the 5S rDNA and the method only identify Candida albicans and detect other Candida species without identifying each organism. Holmes and MyWorld both use universal primers that amplify 18S rDNA from several fungi, including Candida species, Aspergillus fumigatus, Cryptococcus neoformans and Trichosporon species. The amplicons are hydrolyzed with restriction enzymes, and the cleaved fragments are sorted by size by gel1-electrophoresis. The polymorphism aspect of the restriction frement length makes it possible to identify most organisms, but not all organisms. The technique can be used for DNA amplified from pure culture. Since clinical specimens, such as saliva, generally contain a variety of fungal organisms, the technique is of little practical use for diagnostic purposes, since many overlapping fragments obtained from a mixture of fungi will be almost impossible to interpret. The lot uses the above 5.8S and internally transcribed spacers (ITS2) to identify and differentiate Candida albicans and similar Candida species. Makimura amplifies 687 base pair fragments from 25 medically important fungal 18S rDNAs and uses the fragments for the diagnosis of Candida albicans in clinical samples. Mitchell uses nested PCR (nested PCR) to amplify 5.8S, and uses internally transcribed spacers (ITS) to identify Cryptococcus neoformans. There was no subsequent test to verify the identification of the amplified DNA. Nakamura uses 18S primers to detect Aspergillus fumigatus infections in the lungs. Most of the methods given in the above references can only be used to detect an extremely limited number of fungi from clinical specimens. Hopper and MyWorld can detect a variety of organisms from pure culture, but their usefulness for clinical specimens containing a variety of fungal species is at best limited.

Weisburg et. Al. Have registered a US patent for improved markers for detecting small subunit ribosomal RNA sequences of 18S in fungi. The label will detect fungi from many species, but cannot be used to easily identify any single species. Milliman has also registered a United States patent for improved markers for the specific detection of Staphylococcus aureus bacteria based on 16S ribosomal sequences. Hogan et. Al., European Patent Application No. 0,272,009, discloses one bacterial marker for 18S rRNA and three bacterial markers for 28S rRNA sequence. Two of the 28S rRNA labels detect slightly different fungi, while the third label detects Candida krusei from a limited panel of ten bacteria. None of the 28S labels described by the liver is related to the labels described herein. All markers claimed in the present invention can be placed within the first 900 base pairs of the 28S gene. The label initiated by the liver is placed at least 3 ′ on the 28S sequence between base pair 1000 and 2000 (the number is the primary sequence of the Saccharomyces cerevisiae 28S rRNA gene. GenBank score number: J01355). . Leclerc et. Al. Published a report analyzing phylogenetic relationships between fungi based on the partial DNA sequence of some of the 28S genes sequenced by them. Some of the above organisms claimed to be sequenced by Rucklock are identical to some organisms sequenced by us. The organisms are Sporothrix schenckii, Pseudallescheria boydii, Blastomyces dermatitidis, Histoplasma capsulatum and Chrysosporium species. Luklock did not disclose any sequence data in their report, and to our knowledge, they did not make the sequence publicly available in the gene bank. The reverse-complement sequence of their sequencing primer 401 (TCCCTTTCAA CAATTTCACG) overlaps our SEQ ID NO: 1 (GTGAAATTGT TGAAAGGGAA) and 19 nucleotides, and their sequencing primer 636 (GGTCCGTGTT TCAAGACGG) is our SEQ. ID NO: 2 (GACTCCTTGG TCCGTGTT) overlaps with 10 nucleotides. We know that there are no reports of studies of variable regions from the fungus 28S rRNA gene, which has been used as a target for the development of species-specific diagnostic markers.

As discussed above, most existing techniques for molecular detection of fungi rely on the use of very specific primers for PCR amplification of only one fungal species. Using pluripotent primers for PCR amplification of DNA isolated from various organisms utilizes a pre-PCR amplicon identification technique that is useful only in pure cultures of fungi. The technique is unable to identify fungi from clinical specimens containing various fungal organisms. The first purpose of this balming was to develop pluripotent primers for the 28S gene. The primer will be able to amplify in PCR the 28Sr RNA isolated from most fungi. Another object of the present invention was to develop a species specific probe for the fungus of interest, which would be used to analyze the pluripotent 28S amplicon of the present invention. The species specific marker will be able to detect the presence of the fungus of interest even in situations containing mixed fungal species.

One object of the present invention is to provide nucleic acid primers capable of detecting 28S sequences from most fungi of DNA or RNA. Such primers will be used as pluripotent primers in polymerase chain reactions to amplify 28S sequences from any bacteria present in clinical, food, environmental or other samples. In addition, the pluripotent primers will be used to sequence heavy DNA. The sequence obtained will be used to identify the fungus by comparison with a database of known fungal sequences.

The second object of the present invention is the pathogens Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, cryptococcus neoformans, Histoplasma capsulatum, Aspergillus flavus, aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Candi Candida dravilierida, Candidida dabrabrayrii , Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Pseudallescheria boydii, Sporothrix schenckii and other species using any of several other formats, to provide a nucleic acid label that can be detected and confirmed by nucleic acid hybridization. Furthermore, nucleotide sequence information is provided to identify these pathogens and other fungi by DNA sequence comparison (FIG. 2) or by construction of additional markers.

[Background]

Nucleic acid markers and primers are described for detecting fungi that cause disease in humans and animals as well as food and beverage rot. The label can detect rRNA, rDNA or polymerase chain reaction products from most fungi in clinical, environmental or food samples. Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, cryptococcus neoformans, Histoplasma capsulatum, Aspergillus flavus, aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Candida glabrata, Candida guillierp Nucleic acid hybridization markers specific for tropicalis, Pseudallescheria boydii, Sporothrix schenckii and other species (Tables 1 and 2) are described.

Detailed description of the invention

The first object of the present invention was to develop nucleic acid primers for use in polymerase chain reactions that amplify 28S genes from all fungi that are likely to be present in clinical specimens. The amplified DNA can eventually be labeled and analyzed with several other species specific labels. Under hybridization conditions, each of these species specific markers is attached to 28S ribosomal DNA isolated from only one strain, thereby detecting and identifying the species present in the clinical specimen. The 28S gene was chosen as the target because the gene has a region that is conserved between fungi, and the region provides a strong annealing site for pluripotent bacterial markers. In addition, the ribosomal 28S gene was expected to have a hypervariable region unique enough to provide a site for species specific markers. The large rRNA gene refers to the 23S rRNA gene in prokaryotes, and the 28S gene in eukaryotes. The definition is based on length and consequently on the sedimentation coefficient of the rRNA molecule. Fungal large subunit rRNAs vary in size among different organisms and are often considered to be 25S, 26S or 28S. The fungus is a eukaryotes, and to maintain consistency in this application, the large subunit rRNA of the fungus is referred to as 28S rRNA.

The sequences published from Cryptococcus neoformans, two Candida albicans, Saccharomyces cerevisiae and two Schizosaccharomyces pombe 28S electrons are about 3.5 kilobases (kb) in length (GenBank Accession Numbers: L14068, L28817, X70659, J01355, Z19136 Z19578). The four sequences were arranged and regions of sequence variability were found dense between coordinates 200 and 700 from the 5 'end of the gene. As a starting point, two nucleic acid primers P1 (ATCAATAAGC GGAGGAAAAG) and P2 (CTCTGGCTTC ACCCTATTC) (which can hybridize to all four organisms mentioned above and which cannot hybridize to the human 28S sequence (GenBank Accession Number: M11167)) 1) was designed and used in the polymerase chain reaction under low stringency hybridization conditions, the polymerase chain reaction was applied to the hypervariable region in the following 34 fungi obtained by the collection of the Mayo Clinic bacteria. It amplifies about 800 base pairs of DNA: Acremonium species, Aspergillus clavatus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergillus ustus, Beauveria species Blas, Bipolaris, Bipolaris, Bipolars albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida kursei, Candida lusitaniae, Candida parapsilo sis, Candida tropicalis, Chrysosporium species, Cladosporium species, Coccidioides immitis, Cryptococcus neoformans serotype A, Curvularia species, Geotirchum species, Histoplasma capsulatum, Mucor species, Penicillium species, Pseudallescheria boydii, Saccharomyces cerevisiaron Sporothrix trichos.

DNA was extracted from the fungi listed above by the following method. One loop of bacterial culture was scraped from the culture plate using a sterile inoculation loop. The bacteria were added to 1 ml sterile water in a 1.5 ml Sarsted (Newton, North Carolina) screw cap micro-centrifuge tube. In order to lyse the bacteria and elute DNA from the cells, the tube was left for 20 minutes in a boiling water bath. 2 μl of the total cell lysate was used to amplify 28S rDNA in PCR. All PCR amplifications were performed using hot-start using a Perkin-Elmer (Norwalk, CT) 0.5 ml thin membrane polypropylene tube and a Perkin-Elmer thermal cycler in a 50 μl reaction volume. start) as a reaction. The reaction added to the tube for the first time was 2.5 μl of 10 × PCR buffer (100 mM Tris pH 8.3, 500 mM KC1,15 mM MgC1 ₂ ), 5.0 μl of 50% glycerol / 1/1 cresol red, 8.0 Μl of dNTP mix (1.25 mM each of dATP, dGTP, dTTP and dCTP), 12 picomoles of each nucleic acid primer and 25 μl of sterile water to adjust volume. Wax bubbles (wax bead, Ampliwax Gem-100, Perkin-Elmer) were added and the tube was heated at 77 ° C. for 1 minute and cooled to room temperature to form a wax barrier roll. 2.5 μl 10X PCR buffer, 5.0 μl 50% glycerol / 1 mM cresol red, 0.2 μl taq polymerase (Taq polymerase, AmpliTaq 5U / μl, Perkin-Elmer) and 15.3 μl sterile water were the total cell lysates of the bacteria It was added with 2.0 μl of DNA isolated from. 50 cycles of the thermal circuit were performed for 30 seconds at 94 ° C., 1 minute at 40 ° C., and 2 minutes at 72 ° C. The amplified DNA was electrophoresed and precipitated isopropanol after tris buffered phenol1 pH 8.0, phenol / chloroform / isoamyl alcoho1 (volume ratio 25: 24: 1) and three ether extractions A method and a 70% ethanol wash were performed to separate from the low melting agarose-gel.

Both strands of DNA further amplified from the organisms listed above were fully sequenced. All sequencing was performed on an Applied Biosystems 373A sequence device. All nucleotides in the sequence generated were verified and confirmed by examining complementary nucleotides from the sequence of the second strand. We now have a novel database of nucleic acid sequences spanning the variable region of 28S rDNA from various collections of medically important fungi.

While the complete sequences for the Candian albicans, Cryptococcus neoformans and Saccharomyces cerevisiae 28S genes were previously published and deposited in the gene bank, it is clear that which sequence identical regions between the three organisms will also be conserved among all target fungi. You can't stop it. DNA sequences isolated from all of the fungi in the novel 28S database of the present invention had to be analyzed to develop pluripotent 28S markers. All sequences followed extensive manipulations to identify optimal relative alignments with pluripotent labels to identify similar regions. The selected marker sequence had to offer some important characteristics and suggest the conditions present in the 28S gene isolated from most bacterial species. Each marker sequence required a suitable thermal profile, secondary structure and utility in DNA amplification reactions. The markers were optimized to run PCR amplification in the pure culture of bacteria as well as in the presence of DNA isolated from various sources in the case of clinical specimens. The label was also designed to facilitate direct sequencing of the amplified DNA. Analysis of the present invention has found oligonucleotide markers listed in (SEQ ID NO: 1) and (SEQ ID NO: 2) (see FIG. 2 for their location). Successful identification of these two markers ((SEQ ID N0: 1) and (SEQ ID N0: 2)) is ours to develop nucleic acid markers to hybridize and detect 28S rRNA and rDNA isolated from most of the fungus. The objective was achieved (FIG. 1 and Table 1). As shown later in the present invention, the novel sequence information generated by the use of the pluripotent markers of the present invention is characterized by species-specific markers ((SEQ ID NO: 3) that can identify 19 different disease-causing organisms, and (SEQ ID NO: 23).

Table 1a

Presence of hybridization positions for the markers SEQ ID NO: 1 and SEQ ID NO: 2 in the 28S nucleic acid sequence

Table 1b

Presence of hybridization positions for the markers SEQ ID N0: 1 and SEQ ID NO: 2 in the 28S nucleic acid sequence

The markers SEQ ID NO: 1 and SEQ ID NO: 2 were used to successfully amplify (Table 2) and sequence the sequence DNA (Figure 2) over the variable regions isolated from the following 49 organisms: Acremonium species, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergillus ustus, Beauveria species, Bipolaris species, Blacestomyces decanzi kedis , Candida parapsilosis, Candida tropicalis, Chrysosporium species, Cladosporium species, Coccidioides immitis, Cryptococcus neoformans serotype A, Cryptococcus neoformans var. gattii serotype B, Cryptococcus terreus, Cryptococcus laurentii, Curvularia species, Filobasidiella (Cryptococcus) neoformans var bacillispora serotype C, Filobasidiella (Cryptococcus) neoformans var neoformans serotype D, Filobasidium capsulatum, Filobasidium captumuli species Histoplasma capsulatum, Malbranchea species, Mucor species, Paecilomyces species, Penicillium species, Pseudallescheria boydii, Rhizopus species, Saccharomyces cerevisiae, Scopulariopsis brevicaulis, Scopulariopsis brumptii, Sporothrix schenkii and Trichosporon beigelii.

The list contains all four serotypes (A, B, C and D) of Cryptococcus neoformans. The sequence information obtained by using the markers SEQ ID NO: 1 and SEQ ID NO: 2 expanded the size of the database of the present invention consisting of the 28S sequences of the bacteria. All amplified DNA was sequenced across both strands obtained from at least two different segregation populations of each organism to confirm the accuracy of the data obtained.

Table 2a

Amplification of Polymerase Chain Reaction of 28S rDNA with Labels SEQ ID NO: 1 and SEQ ID NO: 2

Table 2b

Amplification of Polymerase Chain Reaction of 28S rDNA with Labels SEQ ID NO: 1 and SEQ ID NO: 2

The above list of fungi sequenced in the present invention represent organisms responsible for most of subcutaneous and deep fungal infections in humans, and also includes parasites (saprohytes) that are commonly found in clinical sequestration populations. Since the two markers (SEQ ID NO: 1 and SEQ ID NO: 2) are hybridized to 28S rDNA obtained from all the fungi listed above, it is possible to diagnose the presence of most fungi that are likely to be present in clinical samples. The markers are believed to be primers for detecting fungi as a whole.

In addition, the markers listed in SEQ ID NO: 1 and SEQ ID NO: 2 hybridize and amplify 23S sequences from bacteria by examining the hybridization positions between the 23S genes of 539 bacteria registered in the gene bank (polymerase chain reaction). Within their potential). The 23S rDNA of the bacteria does not have the appropriate hybridization positions in SEQ ID NO: 1 and SEQ ID NO: 2, and the two labels should not be able to amplify bacterial DNA under stringent conditions.

The second object of the present invention is Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus flavus, Aspergillus glaucus, Aspergillus niger, Aspergillus terguilda ter Candida, Candida glare, To develop species-specific markers for detecting Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropiclis, Pseudallescheria boydii and Sporothrix schenckii. In the present invention, a database of a 28S nucleic acid sequence of a bacterium that makes a multisequence sequence of all organisms sequenced by the present invention was used. Every single sequence is compared only with every other sequence in our database to find unique regions of the sequence that exist only in the subject group and not in all other fungi. When a specific stretch of sequence was identified, that particular portion was analyzed for thermal profile and secondary structure. Each label prepared in the present invention will specifically hybridize and detect nucleic acid sequences from specific regions of only one particular target organism under hybridization conditions. It will be appreciated that the details of a single stranded DNA sequence include the utility of complementary DNA sequences as well as two equivalent RNA sequences, as mentioned in the above description. Moreover, any portion of the sequence insertion modification, including nucleic acids (ie, bases, sugars or skeletons), is functionally equivalent to the sequence. It should also be appreciated that the additional sequence acts as a label or primer. As a result, what is mentioned in the above technique indicates that comparison of a wide range of DNA sequences provides full variability and uniqueness to differentiate organisms (FIG. 2).

Nucleic acid sequences for these species specific synthetic markers are listed in SEQ ID NO: 3 to SEQ ID NO: 23. The sequence includes two markers specific for Cryptococcus neoformans, two markers specific for Sporothrix schenckii, and Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Histoplasma capsulatum, Aspergillus flavus, Aspergillus nilauter, Aspergillus nilauter, Aspergillus niper, There is one marker specific for 28S rRNA and rDNA of Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis and Pseudallescheria boydii (see Tables 3-6 and the following additional description).

All species specific markers developed in the present invention are novel and, as far as we know, have not been reported in the literature. While all 28S genes sequenced in the present invention have several regions that are analyzed differently from species to species, the regions that best function as species specific markers under hybridization conditions are not apparent. Extensive analysis of each 28S sequence yielded several potential marker positions. The marker sites have been studied in more detail to enable the selection of the optimal specific location for each probe based on the need to obtain optimal hybridization characteristics under test conditions. The very specific hybridization characteristics of all the marker sequences sequenced by us were demonstrated by experimental results. The preceding presence of the sequence for only one serotype of the Candida albicans and Cryptococcus neoformans 28S genes in the gene bank makes it possible to develop species specific markers for one of the two organisms for one individual referred to herein. In essence, it is not enough. Before we can identify potent regions for the development of species-specific markers for these two groups of organisms that do not cross react with the above-listed organisms, Candida albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida drusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans serotype A, Crytococcus neoformans var. The gattii serotype B, Cryptococcus terreus, Cryptococcus laurentii, Filobasidiella (Cryptococcus) neoformans var bacillispora serotype C, Filobasidiella (Cryptococcus) Neoformans var neoformans serotype D, Filobasidium capsuligenum, and Filobasidium uniguttulatum were obtained from new sequences.

Modifications of the Chomczynski method (see Example 2 below) allow DNA sequences to be obtained from clinical samples that are not related to each other (see Table 8 for the diversity of tested clinical samples). PCR amplification and subsequent labeling can be easily completed within 24 hours. As a result, the final confirmation can be made within one day as opposed to the days or weeks required by traditional methods. The speed and sensitivity of the diagnosis affects the lives and deaths of debilitated patients fighting fungal disease due to unidentified causes. Early diagnosis enables patients to direct their prescriptions for treating the causative organisms that are being identified rather than waiting for days or weeks to be controlled by unidentified organisms.

The markers of the present invention have the ability to screen accurate target organisms even in mixed fungal infections because of their high level of specificity. Methods such as Hopper and MyWorld cannot identify individual species in mixed fungal infections, as it is hard to interpret when the restriction fragment length polymorphism results in multiple organisms contributing to the restriction fragment. It is because it is impossible. Their method can, as a result, be used only in pure cultures, which also cannot save diagnostic time since the bacteria must first grow into cultures.

The label developed in the present invention enables the rapid species identification of a large number of pathogens by using multiple labels for only one PCR amplified fragment of DNA. Combined with the modified DNA extraction techniques of the present invention and the ability to accurately diagnose in the case of mixed organisms, the strategy can provide maximum diagnostic information in a minimum amount of time. In addition, the diagnostic strategy can be analyzed by automation, which can result in even more savings in time, cost and effort.

In addition to modifications of the above sequences, the sequences and complementary sequences claimed herein can potentially be used in methods for identification of fungi based on some existing methodologies as well as future enhancements and alterations of the technology. Such techniques include methods based on hybridization, ligation, polymerization, depolynerization, sequencing, chemical degradation, enzymatic hydrolysis, electrophoresis, chromatography and amplification, but It is not limited to a method. Furthermore, all such modifications are ultimately based on some selection or amplification method, some ligands, or portions of some nucleic acids that recognize or utilize the sequences of (SEQ ID NO: 1) to (SEQ ID NO: 23) as claimed herein. . Such modifications can be accomplished using numerous linear or exponential target amplification schemes such as numerous forms of PCR, the ligase chain reaction, Q-beta repliase, and the like (SEQ ID NO: Direct detection of species-specific nucleic acids purified or extracted from pure group culture using a label selected from groups 3) to (SEQ ID NO: 23); complementary to (SEQ ID NO: 1) to (SEQ ID NO: 23) The use of DNA; the use of RNA forms of such sequences and complementary sequences; And the addition of one or more reporter sites from various labels, including nucleic acid sequences, proteins, signaling ligands such as acridinium esters, and / or para1llagnetic particles. The use of derivatives of such DNA or RNA sequences includes, but is not limited to such use. The technique can be used with modified derivatives used as DNA, RNA or target or detection molecules.

In addition to the 23 sequences of SEQ ID NO: 1 to SEQ ID NO: 23, we also describe 51 additional sequences of SEQ ID NO: 24 to SEQ ID NO: 74. The 51 sequences include SEQ ID NOs: 3 to SE.Q ID NOs: 23 and vary in sequence arrangement with coordinate 1 consistent with base number 431 of the control S. cerevisiae 28S rRNA gene. (The number is compared with the primary sequence of the control S. cerevisiae 28S rRNA gene. Gene Accession Number: JO1355). The sequence was obtained by amplifying and sequencing 28S rDNA from various fungi with primers of SEQ ID NO: 1 and SEQ ID NO: 2. (The above SEQ ID NO: 1 is coordinated with 403-422 of control S. cerevisiae. And SEQ ID NO: 2 is consistent with coordination 645-662 of control S. cerevisiae).

Analysis of the arranged sequences led to the development of the species specific markers SEQ ID NO: 3 to SEQ ID NO: 23, wherein the label positions are underlined. The 51 arranged sequences include a variety sufficient for those skilled in the art to develop species specific hybrid markers from 10 to 50 nucleotides in length. Similarly, longer species specific hybridization markers comprising the entire 200+ nucleotide length can also be designed. Identification can also be performed by direct DNA sequencing of any DNA amplified with primers SEQ ID NO: 1 and SEQ ID NO: 2. If the derived sequence is about 98% or more identical to any sequence in SEQ ID NO: 24 to SEQ ID NO: 74, the identity of the organism may be identified.

Moreover, we know that portions of SEQ ID NO: 24 to SEQ ID NO: 74 are specific for a group of fungi that are phylogenetically aligned at the genus or above. In addition, SEQ ID NO: 24 to SEQ ID NO: 74, their complementary sequences, together with modifications of the above sequences, are present in existing methodologies and future techniques as described above for SEQ ID NO: 1 to SEQ ID NO: 23. It will be very useful for the investigation to identify fungi based.

Description of Figure 2:

The multiple sequence arrangement shows the sequence of 28S ribosomal RNA gene amplified with primers SEQ ID NO: 1 and SEQ ID NO: 2. 21 species specific markers (SEQ ID NO: 3 to SEQ ID NO: 23) are underlined. Few sequence modifications between two isolates of the same organic agent are indicated by the appropriate code (see abbreviations below). The main difference between the Rhizopus species is indicated by the inclusion of three separate Rhizopus sequences in the array (the organisms in this figure are listed according to their sequence relevance).

Symbol abbreviation:

(.) Spacing to facilitate alignment within the sequence

(R) A or G

(W) A or T

(Y) T or C

(M) A or C

(K) T or G

(S) G or C

(B) T, G or C

Further modifications of the invention using the named sequences will be apparent to those skilled in the art. The following examples illustrate various objects of the present invention, but do not limit its usefulness.

[Brief Description of Drawings]

1 shows the relative position of the sequence recorded on the 28S subunit of the fungus.

2A, B, C and D show various sequence alignments all over (SEQ ID NO: 24) to (SEQ ID NO: 74).

EXAMPLE

Example 1

Test for Hybridization Specificity of Markers SEQ ID NO: 3 to SEQ ID NO: 23

The labels listed in SEQ ID NO: 3 or Q ID NO: 23 were tested for specificity for their target organisms. The label SEQ ID NO: 5 of Candida albicans was first tested against a panel of fungi obtained from the myo clinic collection. Aspergillus species, Aspergillus clatus, Aspergillus clavatus, Aspergillus flavus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergillus ustus, Aspergillus, , Candida guilliermondii, Candida kefyr, Candida Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Chrysosporium species, Cladosporium species, Coccidioides immitis, Cryptococcus neoformans serotype A, Curvularia species, Fusarium species, Geotrichum species, Histoplasma capsula Penicula species 28S rDNA obtained from Pseudallescheria boydii, Rhizopus species, Saccharomyces cerevisiae, Scopulariopsis brevicaulis, Sporothrix schenkii and Trichosporon beigelii are amplified in polymerase chain reaction using oligonucleotide markers of SEQ ID NO: 1 and SEQ ID NO: 2. All PCR amplifications were performed as a hot-start reaction using a Perkin-Elmer (Norwalk, CT) 0.5 ml thin membrane polypropylene tube and a Perkin-Elmer thermal circuit in a 50 μl reaction volume. . The reaction added to the tube for the first time was 2.5 μl of 10 × PCR buffer (100 mM Tris pH 8.3, 500 mM KCl, 15 mM MgCl ₂ ), 5.0 μl of 50% glycerol / 1 mM cresol red, 8.0 μl of dNTP mixture (dATP, dGTP, dTTP And 1.25 mM each of dCTP), 11 picomolar nucleic acid primers and sterile water to adjust volume of 25 μl. Wax bubbles (wax bead, Ampliwax Gem = 100, Perkin-Elmer) were added and the tube was heated at 77 ° C. for 30 seconds and cooled to room temperature forming a wax boundary. 2.5 μl 1OX PCR buffer, 5.0 μl 50% glycerol / 1 mM cresol red, 0.2 μl tar polymerase (Taq po1ymerase, AmpliTaq 5U / 1 μl, Perkin-Elmer) and 15.3 μl sterile water were the total cell lysates of the bacteria It was added with 2.0 μl of DNA extracted from. 50 cycles of the thermal circuit were performed for 30 seconds at 94 ° C., 1 minute at 50 ° C., and 2 minutes at 72 ° C. To visually confirm successful amplification of the 28S rDNA obtained from each of the above listed bacteria, 5 μl of polymerase chain reaction mixture from each sample was run on a 5% polyacrylamide gel. 40 μl of residual amplified 28S rDNA was denatured in 1N NaOH, and half of the denatured rDNA was deposited in a positively charged polysulphone to form an equilibrated membrane in 0.5N NaOH. The membrane was air dried for 15 minutes and baked for 30 minutes in a vacuum oven at 80 ° C. RDNAs amplified from each species were finally bound and fixed at separate points on the membrane. Free binding sites on the membrane were inhibited by incubating the membrane for about 3 hours at 40 ° C. in hybridization buffer (100 ml of the hybridization buffer was 1 g of unexpected milk powder, 6 g of NaH ₂ PO ₄ , 7 g SDS, 0.5 μl of 0.5 μl). Prepared using M EDTA and adjusted to pH 7.2 with NaOH). Prepared using 0.5 M EDTA and adjusted to pH 72 with NaOH). Labels specific for Candida albicans (SEQ ID NO: 5) were labeled terminally with radioactive phosphorus using ³² P ATP and T4 polynucleotide kinases. The label was added to a hybridization buffer of 70 ml of 50 picomolin and the membrane was labeled overnight at 40 ° C. The membranes were washed with immersion buffer at 40 ° C. for 15 minutes and then at 2 × SSC for 15 minutes at 40 ° C. The membrane was exposed on the x-ray film for at least 1 hour after being washed. The oligonucleotide marker SEQ ID NO: 5 is highly specific for Candida albicans. The oligonucleotide marker SEQ ID NO: 5 is distinguished from the sequence of other species of Candida by one or two bases, but this mismatch is sufficient to prevent stable hybridization from forming with other Candida species.

As described above for Candida albicans marker SEQ ID NO: 5, the markers SEQ ID NO: 3 to SEQ ID NO: 23 were tested for specificity for the same panel of fungi listed in the previous paragraph. Candida albicans markers The positively charged polysilphones forming the membrane labeled with SEQ ID NO: 5 were washed with 0.5N NaOH for 10 minutes at 40 ° C. to remove all surrounding Candida albicans markers. The membrane was labeled continuously using all of the labels listed in SEQ ID NO: 3 to SEQ ID NO: 23. For each successively tested label, the membrane was stopped for at least 30 minutes, label hybridization was performed at 40-42 ° C. for at least 3 hours, and post-hybridization wash was performed at about 20 with 2 × SSC solution. Done for a minute. The membrane was removed between the labels by washing with 0.5-1.0 N NaOH solution at 40-42 ° C. The results are listed in Tables 3-6.

As shown in Tables 3 to 6, each label listed in SEQ ID NO: 3 to SEQ ID NO: 23 is specifically hybridized to the 28S nucleic acid sequence of only one target bacterium. This specificity is essential for identifying bacteria of a given species in clinical specimens containing mixed group organisms with high levels of reliability. The 39 organisms listed in the table above represent most organisms commonly isolated from clinical specimens. While we are developing 21 species specific markers (SEQ ID NO: 3 to SEQ ID NO: 23) that identify all 19 individual organisms, the markers of the present invention are unlike any other fungi that are likely to be present in clinical samples. Additional organisms listed in the test panel were used to confirm that they also had no cross-linkability. The ability to diagnose accurately and reliably and identify the large number of pathogens at the species level cannot be countered by any other report. The fact that we can obtain this by labeling DNA amplified by a pair of pluripotent markers (SEQ ID NO: 3 to SEQ ID NO: 2) is based on 21 different markers (SEQ ID NO: 3 to SEQ ID There is a number of advantages in that diagnostics save time, money and effort by providing the ability to test a single amplified target with NO: 23.

Gene bank investigations were performed using all of the labels listed in SEQ ID NO: 3 to SEQ ID NO: 23 to determine whether similar sequences exist in the database. The 28S sequences for one serotype of Candida albican and Cryptococcus neoformans already exist in the gene bank, and as expected, the markers for Candida albicans and Cryptococcus neoformans exactly matched the 28S sequences isolated from the two organisms. Ten other markers also corresponded to DNA isolated from various genera not related to the 28S gene (Table 7). This was expected because the identity of short stretched sequences can often be found for query sequences in unrelated genes isolated from the same or different organisms. This observation is known to be mentioned in the art. In all cases, the sequence corresponding to the marker sequence was not located within the 28S rRNA gene. The label of the invention is used to analyze 28S DNA previously amplified in a polymerase chain reaction with the labels SEQ ID NO: 1 and SEQ ID NO: 2. Under stringent conditions, the two labels amplify DNA from the group of 28S rRNA genes. Therefore, DNA that has not been amplified from the non-28S genes listed in Table 7 will be useful for the hybridization of the markers SEQ ID NO: 3 to SEQ ID NO: 23. The presence of related genes in the non-28S, i.e., non-amplified genes, will not be detected, and consequently will not have any effect on the sensitivity or specificity of the detection and identification strategies of the present invention.

Table 3a

Detection of Species Specific 28S Sequences Using Markers SEQ ID NO: 3 to SEQ ID NO: 8

Table 3b

Detection of Species Specific 28S Sequences Using Markers SEQ ID N0: 3 to SEQ ID N0: 8

Table 4a

Detection of Species Specific 28S Sequences Using Markers SEQ ID NO: 9 to SEQ ID NO: 14

Table 4b

Detection of Species Specific 28S Sequences Using Markers SEQ ID N0: 9 to SEQ ID N0: 14

Table 5a

Detection of Species Specific 28S Sequences Using Markers SEQ ID NO: 15 to SEQ ID NO: 20

Table 5b

Detection of Species Specific 28S Sequences Using Markers SEQ ID N0: 15 to SEQ ID N0: 20

Table 6a

Detection of Species Specific 28S Sequences Using Markers SEQ ID NO: 21 to SEQ ID NO: 23

Table 6b

Detection of Species Specific 28S Sequences Using Markers SEQ ID NO: 21 to SEQ ID NO: 23

Table 7a

Genetic bank survey listing genes isolated from other organisms having 100% identity with markers SEQ ID NO: 3 to SEQ ID NO: 23

Table 7b

Genetic bank findings listing genes isolated from other organisms having 100% identity to the labels SEQ ID NO: 3 to SEQ ID NO: 23

* Note: As described herein above, the presence of sequences similar to labels SEQ ID NO: 3 to SEQ ID NO: 23 in genes not related to 28S have no effect on the specificity or sensitivity of the diagnostic strategy of the present invention. Not crazy Species specific markers of the invention are used to analyze 28S DNA previously amplified in a polymerase chain reaction using the markers SEQ ID N0: 1 and SEQ ID N0: 2 of the invention. The two markers do not amplify the DNA of any gene in the 28S object in column # 4 (comparable gene), therefore any DNA amplified from the non-28S gene is labeled with SEQ ID NO: 3 to SEQ ID NO. Not useful for hybridization of: 23.

Example 2

Use of the method of Example 1 to test a clinical specimen for a particular bacterial organism

Clinical specimens obtained from the respiratory tract and esophagus of healthy people almost always contain some flora. Most of these bacteria are non-pathogens, but may also show inaccurate positive responses to traditional immunochemical diagnostic tests against pathogens.

We obtained 44 clinical samples from various sources spanning intervertebrak discs and lung biopsies in saliva and incision drainage tubes. Typical specimens and culture results showed that all 44 specimens contained at least one form of bacteria. To test the efficiency of the label of the present invention, we extracted DM from all 44 samples and labeled SEQ ID N0: 1 2 in the polymerase chain reaction to amplify the 28S sequence of the bacteria present in the sample. Was used.

Modified Chomczynski and Sacchi techniques, which first described the use of acid guanidinium thiocyanate-pheno1-chloroform to preferentially extract RNA from cells and tissues. DNA was extracted from all clinical samples. In order to preferentially extract DNA from cells, we actually replaced the cell temperature lysis with boiling lysis, and the acid guanidinium thiocyanate-phenol-chloroform was replaced with alkaline phenol. -Substituted with guanidine thiocyanate. 1.5 μl of Sarsted (Newton, North Carolina) polypropylene screw cap tubes with 0-ring sutures were used for extraction. 200 μl of sample was added to 500 μl of GTP reagent (guanidine thiocyanate 6M dissolved in 50 mM Tris pH 8.3 solution mixed with equal volume of phenol buffered at Tris pH 8.0). The solution was mixed by stirring and immediately left for 15 minutes in a boiling water bath. The tube was run in a microcentrifuge for 5 seconds and 250 μl of chloroform / isoamyl alcohol solution (volume ratio 24: 1) was added and stirred to mix. The solution was centrifuged for 10 minutes to separate the liquid phase and 450 μl of aqueous solution (top) was transferred to a new tube. The aqueous phase was mixed with 500 μl of 100% isopropane and left at -20 ° C. for at least 1 hour. The tube was centrifuged for 15 minutes at almost the end of the standing time and the supernatant was removed without disturbing the nucleic acid precipitate. The precipitate was washed by gently shaking up and down twice with 500 μl of ice cooled 70% ethane solution to remove the remaining portion of the GTP reagent. The ethanol was removed and the precipitate was dried for 10 minutes in a speed vac oven. The precipitate was re-clouded with 25 μl of sterile deionized distilled water and 5 (μl) of the turbid solution was used for 50 (μl) PCR amplification. The PCR was performed in a heat-initiated reaction using thermal circuit conditions for the labels SEQ ID NO: 1 and SEQ ID NO: 2 described in Example 1. Gel-electrophoresis showed that the markers SEA ID NO: 1 and SEQ ID NO: 2 successfully amplified the isolated DNA from all 44 samples.

DNA amplified from each sample was transferred to the polysilphone charged in the amount of the membrane. We radiolabeled the species specific markers SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7 of the present invention and isolated from Aspergillus fumigatus, Candida albicans, Coccidioides and Cryptococcus neoformans The membranes were labeled continuously to test separately for the presence of 28S rDNA. Membrane arrest, label hybridization and washing were done as described in Example 1. The results are shown in Table 8.

In the tested clinical specimens, no inaccurate positive responses were observed and showed 100% specificity for the four labels. Ten of 12 culture positive samples were identified for Aspergillus fumigatus, and 11 of 13 samples were detected for Candida albicans, indicating detection sensitivity of about 85% for the two markers. Furthermore, two of the two Coccidioides immitis and two of the two Cryptococcus neformans were correctly identified (100% detection sensitivity).

As indicated by the above results, the labels described herein can be used in various clinical specimens with high efficiency.

Table 8a

Detection of Aspergillus fumigatus, Candida albicans, Coccidioides immitis and Cryptococcus neoformans Using Species-Specific Markers in Clinical Specimens

Table 8b

Detection of Aspergillus fumigatus, Candida albicans, Coccidioides immitis and Cryptociccus neoformans Using Species-Specific Markers in Clinical Specimens

Example 3

DNA sequence based on identification of unknown bacterial organisms

Another usefulness of the label of the present invention is its rapid DNA sequence based on the identification of the pure culture of the bacteria. The labels SEQ ID N0: 1 and SEQ ID N0: 2 are used to amplify the unknown bacteria 28S rDNA in the polymerase chain reaction. The label SEQ ID N0: 1 or SEQ ID NO: 2 is subsequently used as a sequencing primer to obtain a DNA sequence from the 28S rDNA amplified belonging to the unknown bacterium. The DNA sequence is compared with the 28S DNA sequences of the group shown in the databases herein, and sequence matching or amplifying any of the label sequences in SEQ ID N0: 3 to SEQ ID N0: 74 is You will be sure of the identification of the bacteria. Since the technique contains DNA isolated from more than one bacterium, it cannot be used directly in clinical specimens, and the resulting DNA sequence will consist of duplicate sequences of several organisms. The technique is used to quickly and reliably identify colonies (co1onies) of one bacterium on culture plates, clinical specimens, food, medicinal, environmental, or other specimens containing only one strain.

Example 4

Capture and Identification of Target DNA or RNA

All primers and labels described in the specification of the present invention include, but are not limited to, any detectable radioisotopes, enzymes, antigens, antibodies, chemiluminescent and fluorescent reagents (fluorescent chemica1). It may be labeled as a reporter or signal part. Furthermore, the labels can be modified without altering their desired material by the final addition of nucleotides designed to be inserted at restriction sites or other useful sequences. In addition, the label may be modified by the addition of capture moieties (including but not limited to paramagnetic particles, biotin, fluorescein, didxigenin, antigens, antibodies), The label may be attached to the wall of a microtiter tray to aid in the solid phase capture and purification of the DNA and any DNA or RNA hybridized to the label. The fluorescein may be used as the signal portion as well as the capture portion, and the capture portion is used by interacting with an anti-fluorescein antibody.

Typical uses of this variant are as follows. Primers SEQ ID NO: 1 and SEQ ID NO: 2 will be put to practical use to amplify 28S rDNA from a sample as previously described. Primers will be modified to contain the biotin moiety at their 5 'end. A streptavidin solid phase, such as paramagnetic particles, will be used to separate the PCR product from the reaction mixture. The amplified target is subsequently hybridized to a third label (SEQ ID N0: 3 to SEQ ID N0: 74 or their complementary sequence) attached to a detectable moiety that determines which species of bacteria is present in the given sample. Various labels, each labeled with different detectable moieties, can be used simultaneously to analyze the amplified target.

Optionally, the labels SEQ ID N0: 1 and SEQ ID N0: 2 will be used to amplify 28S rDNA from the sample, as described above. In a separation reaction, independently, one of SEQ ID N0: 1 or SEQ ID N0: 2 will be modified by attaching to a solid phase capture moiety such as a paramagnetic particle, SEQ ID NO: 3 to SEQ ID N0; 74 (or Their complementary sequences) will be modified by the addition of a detectable moiety. Optionally, within said amplicon, any sequence defined by SEQ ID NO: 1 and SEQ ID NO: 74 includes, but is not limited to, SEQ ID NO: 3 to SEQ ID NO: 74. It can be used to. One of said labels attached to a solid phase (SEQ ID N0: 1 and SEQ ID N0: 2) or any other suitably designed sequence and detectable moiety (SEQ ID NO: 3 to SEQ ID NO: 74 or their complementary sequences) One of the markers, modified by attachment to, will hybridize together in the PCR product if in solution, if they are produced. The hybrid, if present, will be captured from the solution and analyzed by a method suitable for the detectable moiety. Detection of the hybridized label will indicate which bacterial species are present in a given sample. Multiple labels, each labeled with various detectable moieties, can be used simultaneously to analyze the amplified target.

Example 5

Species-specific Amplification of Bacillus DM

Another usefulness of the labels described herein lies in their use as primers in the direct detection of certain bacterial species by nucleic acid amplification reactions. In this specific embodiment, one primer is a pluripotent primer such as (SEQ ID NO: 1) or (SEQ ID NO: 2), and the other is (SEQ ID NO: 3) to (SEQ ID NO: 23) Or a species-specific primer selected from the above bacteria consisting of their complementary sequences. One variation of this approach is the substitution of (SEQ ID NO: 1) or (SEQ ID NO: 2) with any functional sequence located proximate to the species-specific primer. Another variation of this approach is the selection of any suitable species specific primer pairs of SEQ ID NO: 24 to SEQ ID NO: 74.

Sequence list

(1) General Information

(i) Applicant:

(A) Sandhugh, Gulfrit S.

(B) Cline, Bruce.

(ii) Name of the Invention: Nucleic Acid Labeler for Detection and Identification of Fungi

(iii) number of sequences: 23

(iv) Payee Address:

(A) Recipient: Shiba Koning Diagnostics Corporation

(B) Street: Notsu Street 63

(C) Poetry: Medfield

(D) Note: Massachusetts

(D country: United States of America

(F) ZIP code: 02052

(v) computer readable form:

(A) Media Type: Disk 3.5 inch, 1.44Mb storage

(B) Computer: IBM PS / 2

(C) operating system: MS-DOS 6.2

(D) Software: Word 6.0

(vi) Current Application Data:

(A) Application number:

(B) filing date:

(C) Classification:

(vii) Preliminary Application Data:

(viii) Patent Attorney Information:

(A) Name: Mogenstern, Arthur S.

(B) registration number: 28,244

(C) Case number: CCD-180

(ix) Communications Information:

(A) Telephone: 508-359-3836

(B) Fax: 508-359-3885

(2) Information about SEQ ID N0: 1:

(i) Sequence Features:

(A) Length: 20

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Labels for Bacterial Organisms

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 1

(3) Information about SEQ ID NO: 2:

(i) Sequence Features:

(A) length: 18

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Markers for Group Organisms

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 2

(4) Information about SEQ ID N0: 3:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Marker for Aspergillus fumigatus

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 3

(5) Information about SEQ ID N0: 4:

(i) Sequence Features:

(A) length: 13

(B) type: nucleic acid

) Number of chains: 1 print

A) Topology: straight

ii) Molecular type: nucleic acid marker for Blastomyces dermatitidis

iii) Estimated Sequence: No

iv) Antisense: No

SEQ ID NO: 4

(6) Information about SEQ ID N0: 5:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Labeler for Candida albicans

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 5

(7) Information about SEQ ID N0: 6:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Markers for Coccidioides immitis

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 6

(8) Information about SEQ ID N0: 7:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Labeler for Cryptococcus neoformans

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 7

(9) Information about SEQ ID NO: 8:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Labeler for Cryptococcus neoformans

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 8

(10) Information about SEQ ID N0: 9:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Labeler for Histoplasma capsulatum

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 9

(11) Information about SEQ ID NO: 1O:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Labeler for Aspergillus glaucus

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 10

(12) Information about SEQ ID NO: 11:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) the number of: 1: 1 print

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Labeler for Aspergillus niger

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 11

(13) Information about SEQ ID NO: 12:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Marker for Aspergillus terreus

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 12

(14) Information about SEQ ID NO: 13:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Labeler for Candida glabrata

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 13

(15) Information about SEQ ID NO: 14:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Nucleic acid marker for Candida guilliermondii

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 14

(16) Information about SEQ ID N0: 15:

(i) Sequence Features:

(A) Length: 14

(B) Type: Hexane

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Labeler for Candida kefyr

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 15

(17) Information about SEQ ID N0: 16:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Labeler for Candida krusei

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 16

(18) Information about SEQ ID N0: 17:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Labeler for Candida lusitaniae

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 17

(19) Get the information for SEQ ID NO: 18:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Markers for Candida parapsilosis

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 18

(20) Information about SEQ ID N0: 19:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Nucleic acid marker for Candida tropicalis

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 19

(21) Information about SEQ ID NO: 20:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Labeler for Pseudallescheria boydii

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 20

(22) Information about SEQ ID NO: 21:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Nucleic acid marker for Aspergillus flavus

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 21

(23) Information about SEQ ID NO: 22:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Nucleic acid marker for Sporrothrix schenckii

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 22

(24) Information about SEQ ID N0: 23:

(i) Sequence Features:

(A) Length: 14

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Nucleic Acid Labeler for Sporthrix schenckii

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 23

(25) Information about SEQ ID N0: 24:

(i) Sequence Features:

(A) length: 208

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) molecular type: Acremonium species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 24

(26) Information about SEQ ID NO: 25:

(i) Sequence Features:

(A) length: 212

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) molecular type: Aspergillus clavatus species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 25

(27) Information about SEQ ID N0: 26:

(i) Sequence Features:

(A) length: 212

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Aspergillus flavus species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 26

(28) Information about SEQ ID NO: 27:

(i) Sequence Features:

(A) length: 212

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) molecular type: Aspergillus fumigatus species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 27

(29) Information about SEQ ID NO: 28:

(i) Sequence Features:

(A) length: 212

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Aspergillus glaucus species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 28

(30) Information about SEQ ID NO: 29:

(i) Sequence Features:

(A) length: 213

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) molecular type: Aspergillus nidulans species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 29

(31) Information for SEQ ID NO: 30:

(i) Sequence Features:

(A) length: 212

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) molecular type: Aspergillus niger species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 30

(32) Information about SEQ ID NO: 31:

(i) Sequence Features:

(A) length: 212

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) molecular type: Aspergillus ochraceus species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 31

(33) Information about SEQ ID NO: 32:

(i) Sequence Features:

(A) Length:

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) molecular type: Aspergillus terreus species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 32

(34) Information on SEQ ID NO: 33:

(i) Sequence Features:

(A) length: 213

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) molecular type: Aspergillus unguis species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 33

(35) Information about SEQ ID N0: 34:

(i) Sequence Features:

(A) length: 212

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) molecular type: Aspergillus ustus species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 34

(36) Information about SEQ ID NO: 35:

(i) Sequence Features:

(A) length: 208

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Beauveria species specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 35

(37) Information about SEQ ID N0: 36:

(i) Sequence Features:

(A) length: 213

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Bipolaris species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 36

(38) Information about SEQ ID N0: 37:

(i) Sequence Features:

(A) length: 105

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Blastoschizomyces species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 37

(39) Information on SEQ ID NO: 38:

(i) Sequence Features:

(A) length: 214

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: B1astomuces dermatitidis species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 38

(40) Information about SEQ ID NO: 39:

(i) Sequence Features:

(A) length: 213

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) molecular type: Chrypsosporium species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 39

(41) Information about SEQ ID N0: 40:

(i) Sequence Features:

(A) length: 207

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Cladosporium species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 40

(42) Information about SEQ ID NO: 41:

(i) Sequence Features:

(A) length: 213

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Curvularia species specific region of 28S gene

(iii) Estimate: No

(iv) Antisense: No

SEQ ID NO: 41

(43) Information about SEQ ID NO: 42:

(i) Sequence Features:

(A) length: 213

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Candida albicans species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 42

(44) Information about SEQ ID N0: 43:

(i) Sequence Features:

(A) length: 223

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Candida g1abrata species-specific region of the 28S gene

(iii) Estimated sequence: No

iv) Antisense: No

SEQ ID NO: 43

(45) Information about SEQ ID N0: 44:

(i) Sequence Features:

(A) length: 212

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Candida guillier'11ondii species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 44

(46) Information about SEQ ID N0: 45:

(i) Sequence Features:

(A) length: 214

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Coccidioides im11litis species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 45

(47) Information about SEQ ID N0: 46:

(i) Sequence Features:

(A) length: 187

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Candidakefyr Species Specific Region of the 28S Gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 46

(48) Information about SEQ ID NO: 47:

(i) Sequence Features:

(A) length: 213

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Candidalkrusei Species Specific Region of the 28S Gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 47

(49) Information about SEQ ID N0: 48:

(i) Sequence Features:

(A) length: 236

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Cryptococcus laurentii species specific region of 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 48

(50) Information about SEQ ID NO: 49:

(i) Sequence Characterization:

(A) Length: 173

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Candida lusitaniae species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Entense: No

SEQ ID NO: 49

(51) Information about SEQ lD NO: 50:

(i) Sequence Features:

(A) length: 238

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Cryptococcus neoformans var gattii (serum type B) species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 50

(52) Information about SEQ ID N0: 51:

(i) Sequence Features:

(A) length: 238

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Cryptococcus neoformans var gattii (serum type B) species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 51

(53) Information about SEQ ID NO: 52:

(i) Sequence Features:

(A) Length: 2l1

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Candida parapsilosis species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 52

(54) Information about SEQ ID N0: 53:

(i) Sequence Features:

(A) length: 238

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Cryptococcus terreus species specific region of 28S gene

(iii) Estimated sequence: No

(iv) Entense: No

SEQ ID NO: 53

(55) Information about SEQ ID NO: 54:

(i) Sequence Features:

(A) length: 211

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Candida tropicalis species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 54

(56) Information about SEQ ID N0: 55:

(i) Sequence Features:

(A) length: 211

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Fusarium species specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 55

(57) Get information on SEQ ID N0: 56:

(i) Sequence Features:

(A) length: 238

(B) Type 1: Nucleic Acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Filobasidiumlcapsuligenum species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 56

(58) Information on SEQ ID NO: 57:

(i) Sequence Features:

(A) Length: 238

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Filobasidiella neoforlnans varbacillispora (serum type C) species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 57

(59) Information about SEQ ID N0: 58:

(i) Sequence Features:

(A) length: 238

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Filobasidiella neoformans varneoformans (serum type D) species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 58

(60) Information about SEQ ID N0: 59:

(i) Sequence Features:

(A) length: 236

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Filobasidium uniguttulatum species specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 59

(61) Information about SEQ ID NO: 60:

(i) Sequence Features:

(A) Length: 204

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Geotrichum species specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 60

(62) Information about SEQ ID N0: 61:

(i) Sequence Features:

(A) length: 214

(B) polyps: nucleic acid

(C) Number of chains: 1 chain

(D) topology: straight

(ii) Molecular type: Histoplasnla capfulatum song-specific inverse of 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 61

(63) Information about SEQ ID NO: 62:

(i) Sequence Features:

(A) length: 215

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Malbtanchea species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 62

(64) Information about SEQ ID NO: 63:

(i) Sequence Features:

(A) length: 237

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Mucor species specific region of 28S gene

(iii) Estimated sequence: No

(iv) Entense: No

SEQ ID NO: 63

(65) Information about SEQ ID NO: 64:

(i) Sequence Features:

(A) length: 209

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Paecilomyces species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 64

(66) Information about SEQ ID NO: 65:

(i) Sequence Features:

(A) length: 199

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Penicillium Species Specific Region of the 28S Gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 65

(67) Information about SEQ ID NO: 66:

(i) Sequence Features:

(A) length: 210

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) molecular type: Pseudallescheria boydii species-specific region of the 28S gene

(iii) Presumption: No

(iv) Antisense: No

SEQ ID NO: 66

(68) Information about SEQ ID NO: 67:

(i) Sequence Features:

(A) Length: 244

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular Type: Rhizopus spp. (NO: 1) specific phenotype of 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 67

(69) Information about SEQ ID NO: 68:

(i) Sequence Features:

(A) length: 215

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Rhizopus species (NO: 2) specific region of 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 68

(70) Information on EQ ID NO: 69:

(i) Sequence Features:

(A) length: 215

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Rhizopus species (NO: 3) specific region of the molecular type: 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 69

(71) Information about SEQ ID NO: 70:

(i) Sequence Features:

(A) length: 210

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Sporothrix species specific region of 28S gene

(iii) Estimated sequence: No

(iv) Entense: No

SEQ ID NO: 70

(72) Information about SEQ ID NO: 71:

(i) Sequence Features:

(A) length: 208

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Scopulariopsis brevicaulis species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 71

(73) Information about SEQ ID NO: 72:

(i) Sequence Features:

(A) length: 210

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) Molecular type: Scopulariopsis brumptii species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 72

(74) Information about SEQ ID NO: 73:

(i) Sequence Features:

(A) length: 214

(B) type: nucleic acid

(C) Number of chains: 1 chain

(D) Topology: straight chain

(ii) molecular type: Sacchlaromyces cerevisiae species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 73

(75) Information about SEQ ID NO: 74:

(i) Sequence Features:

(A) length: 236

(B) type: nucleic acid

(C) Number of chains: 1 chain (D) topology: linear

(ii) Molecular type: Tric11osporon beigelii species-specific region of the 28S gene

(iii) Estimated sequence: No

(iv) Antisense: No

SEQ ID NO: 74

Claims (9)

Acremonium species, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergillus ustus, Beauvaria, Blasto Candida dascalans , Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Chrysosporium spp., Cladosporium spp., Coccidioides immitis, Cryptococcus neoformans var gattii serotype B, Crytococcus neoformans serotype A, Cryptocous terre , Fusarium species, Filobasidium capsuligenum, Filobasidiella (Cryptoccus) neoformans var bacillisopora serotype C, Filobasidiella (Cryptoccus) neoformans var neoformans serotype D, Filobasidium uniguttulatum, Geotrichum species, Histoplasma capsulatum, Malbranche species, Penicillium species Pseudallescheria boydii, Rhizopus species, S Oligonucleotide markers for 28S subunits of the group capable of identifying one species selected from the group consisting of porothrix schenkii, Scopulariopsis brevicaulis, Scopulariopsis brumpti, Saccharomyces cerevisiae and Trichosporon beigelii. The oligonucleotide label according to claim 1, which constitutes all or a part of any one or functionally equivalent sequence of the above sequence from SEQ ID NO: 3 to SEQ ID NO: 74, which can identify the species of interest. sieve. The method according to claim 1, wherein all or part of any one or functionally equivalent sequence of any one or more of the above sequences spanning SEQ ID NO: 74 to SEQ ID NO: 3 capable of identifying one or more of said species of bacteria Oligonucleotide label constituting the. a. Extracting hexane material from the bacteria contained in the sample; b. Adding two known primers (SEQ ID NO 1) and (SEQ ID NO 2), or functionally equivalent primers, which bracket the subject region on the 28S rDNA or rRNA present in the subject group; c. Amplifying the sequences between the primers; And d. Using at least one third labeled label to determine which class of fungus is present, wherein the third label comprises (SEQ ID NO: 3) to (SEQ ID NO: 74), A method for determining whether there is one or more species selected from a group of fungi present in a sample, wherein the sample is selected from the group consisting of any portion and functionally equivalent portions thereof. 5. The method of claim 4, wherein said amplifying procedure is a polymerase chain reaction. The method according to claim 4, wherein after the amplification d. Using two or more third labels to determine what species of said organism is present, wherein one of said third labels is from one or more labels linked to said label and labeled portion And wherein the third label is selected from the group consisting of (SEQ ID NO 3) to (SEQ ID 74), any portion thereof and a functionally equivalent portion thereof. 5. The method of claim 4, wherein the amplifying step is excluded. The method according to claim 4, wherein the species is Acremonium species, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergilla species Blastopolar , Blatomyces dermatitidis, Candida albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Chrysosporium spp., Cladosporium spp., Coccidioides immitis, Cryptococcus neoformus Bactobacillus , Cryptococcus laurentii, Cryptococcus terreus, Curvularia species, Fusarium species, Filobasidium capsuligenum, Filobasidiella (Cryptoccus) neoformans var bacillisopora serotype C, Filobasidiella (Cryptoccus) neoformans var neoformans serotype D, Filobasitodium untrichumula Mucor, Paecilomyces, Penicillum, Pseudallescheria boydii, Rhizopus species, Sporothrix schenkii, Scopulariopsis brevicaulis, Scopulariopsis brumpti, Saccharomyces cerevisiae and Trichosporon beigelii. 5. The method of claim 4, wherein one or more labels are used, wherein each third label is linked to a different signal moiety or to a part that separates from the third label.