# Patent

## Title
Diagnostic assays for the detection and identification of aspergilli

## Abstract

Three important species of Aspergillus, A. fumigatus, A. flavus and A. niger are known to contribute to the pathogenicity of allergic and invasive diseases in humans. They are also known to be plant pathogens. Several important ESTs/genes of Aspergilli species are now identified and characterized. Efforts are still needed to explore 30% genes of Aspergillus species for their valuable products which need to be explored. Polyketide biosynthetic pathway in Aspergillus species produce important secondary metabolites like polyketide toxins such as Aflatoxins, drugs such as Lovastatins and several other important pharmaceutically important polyketide compounds etc. With the availability of Aspergillus genome sequences it is possible today to characterize the structure and function of important genes of Aspergillus species. Based on the gene sequence information on PKS enzymes in medically and agriculturally important Aspergillus species such as A. fumigatus, A. flavus and A. niger sequences of diagnostic use are identified and a multiplex PCR assay is developed using clinical and agricultural samples.

## Background
FIELD OF INVENTION
The present invention relates to PCR based simultaneous detection of A. fumigatus, A. flavus and A. niger in clinical and agricultural samples. The diagnostic assays use specific primers and fluorescent based quantitative PCR assays and facilitate (i) detection of Melanin producing A. fumigatus, Aflatoxin producing A. flavus from A. niger from Agricultural samples and (ii) for specific detection and differentiation of Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger in the clinical samples.
BACKGROUND
Aspergillus species, A. fumigatus, A. flavus and A. niger are the causative agents in human “Aspergillosis” and are also pathogens damaging Agricultural crops. They induce a variety of Aspergillus induced clinical conditions in immunocompetent and immunocompromised hosts. Depending on the host's immunity and the virulence of the clinical spectrum varies from aspergilloma, allergic Aspergillus sinusitis, allergic bronchopulmonary aspergillosis [ABPA], and hypersensitivity pneumonitis and invasive aspergillosis (Agarwal R. Allergic bronchopulmonary aspergillosis. Chest. 2009 March;135(3):805-26). These infections are often and progress fast and eventually fatal in immunocompromised patients. For example, pulmonary and cerebral aspergillosis has mortality rates of 86 and 99% respectively, even when adequately treated (Marr K A, Bowden R A. Fungal infections in patients undergoing blood and marrow transplantation. Transpl Infect Dis. 1999 December; 1(4):237-46). Allergic bronchopulmonary Aspergillosis in immunocompetent persons is often diagnosed by serological tests based on Aspegillus antigens and the specific IgE and IgG antibodies in the serum samples. Serodiagnostic tests based on Antigenic peptides and antigens are reported (Denning D W. Therapeutic outcome in invasive aspergillosis. Clin Infect Dis. 1996 September;23(3):608-15). In case of invasive Aspergillosis, particularly in immunocompromised host, the antibodies are present either negligible quantities or absent. Hence detection of circulating antigen or pathogen in clinical samples is the best strategy. This necessitates the development of dependable, specific methods for the detection of pathogens in clinical samples. In view of this efforts are made to develop more sensitive reagents and protocols for detection of important Aspergillus species in clinical samples.
Gene based methods are reported in the literature for detection of A. fumigatus, A. flavus and A. niger for clinical samples. They are mainly based on ITS regions of Aspergillus species which are genus specific (Abdin M Z, Ahmad M M, Javed S. Advances in molecular detection of Aspergillus: an update. Arch Microbiol. 2010 June;192(6):409-25. Epub 2010 Apr. 1). Further some of the important Aspergillus species such as Aspergillus fumigatus has been reported to develop resistance to Amphotericin and itraconazole. The need for a rapid test to identify Aspergilli to the species level, to assist in the selection of appropriate drugs for the treatment of clinical Aspergillus infections is also of high importance. Nonculture-based methods are increasingly used for rapid, accurate diagnosis to improve the Outcome of patient. New and existing DNA amplification platforms have high sensitivity and specificity for direct detection and identification of fungi in clinical specimens. Novel technologies (e.g., isothermal and PNA FISH methods), platforms enabling high-throughput analyses (e.g., DNA microarrays and Luminex xMAP) and/or commercial PCR assays are some of advances in diagnosis of Aspergillosis (Spiess B, Seifarth W, Hummel M, Frank O, Fabarius A, Zheng C, Mörz H, Hehlmann R, Buchheidt D. DNA microarray-based detection and identification of fungal pathogens in clinical samples from neutropenic patients. J Clin Microbiol. 2007 November;45(11):3743-53. Epub 2007 Aug. 22).
Unique internal transcribed sequence 2 (ITS2) coding regions have been used to develop nucleic acid probes for different species of Aspergillus (A. flavus, A. fumigatus, A. niger, A. terreus, and A. nidulans), as disclosed in U.S. Pat. No. 6,372,430 (U.S. Pat. No. 6,372,430—Nucleic acids for detecting Aspergillus species and other filamentous fungi). Real time PCR methodologies using ITS region in Aspergilli has also been described for specific detection from clinical samples and Agri products (Schabereiter-Gurtner C, Selitsch B, Rotter M L, Hirschl A M, Willinger B Development of novel real-time PCR assays for detection and differentiation of eleven medically important Aspergillus and Candida species in clinical specimens. J Clin Microbiol. 2007 March;45(3):906-14, Ramirez M, Castro C, Palomares J C, Torres M J, Aller A I, Ruiz M, Aznar J, Martín-Mazuelos E. Molecular detection and identification of Aspergillus spp. from clinical samples using real-time PCR.Mycoses. 2009 March;52(2):129-34, Faber J, Moritz N, Henninger N, Zepp F, Knuf M. Rapid detection of common pathogenic Aspergillus species by a novel real-time PCR approach.Mycoses. 2009 May;52(3):228-33, Bolehovska R, Pliskova L, Buchta V, Cerman J, Hamal P. Detection of Aspergillus spp. in biological samples by real-time PCR. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2006 November;150(2):245-8, Mulè G, Susca A, Logrieco A, Stea G, Visconti A.Development of a quantitative real-time PCR assay for the detection of Aspergillus carbonarius in grapes. Int J Food Microbiol. 2006 Sep. 1;111 Suppl 1:S28-34). Recently a high throughput assay based on Luminex xMAP hybridization technology has been described for clinically relevant fungal pathogens including Aspergillus species (Etienne K A, Kano R, Balajee S A. Development and validation of a microsphere-based Luminex assay for rapid identification of clinically relevant aspergilli. J Clin Microbiol. 2009 April;47(4):1096-100). Monochrome LightCycler real-time PCR based on fluorescence probe have also been described for specific quantification of Aspergillus (Bu R, Sathiapalan R K, Ibrahim M M, Al-Mohsen I, Almodavar E, Gutierrez M I, Bhatia K. Monochrome LightCycler PCR assay for detection and quantification of five common species of Candida and Aspergillus. J Med Microbiol. 2005 March;54(Pt 3):243-248, Imhof A, Schaer C, Schoedon G, Schaer D J, Walter R B, Schaffner A, Schneemann M. Rapid detection of pathogenic fungi from clinical specimens using LightCycler real-time fluorescence PCR.Eur J Clin Microbiol Infect Dis. 2003 Sep;22(9):558-60). Some of the methods such as DNA microarrays are also reported for detection of Aspergillus species from clinical samples (Spiess B, Seifarth W, Hummel M, Frank O, Fabarius A, Zheng C, Mörz H, Hehlmann R, Buchheidt D. DNA microarray-based detection and identification of fungal pathogens in clinical samples from neutropenic patients. J Clin Microbiol. 2007 November;45(11):3743-53) but these are expensive to perform and require sophisticated analytical tools to interpret the results. However all these tests are based on internal transcribed sequence and spacer regions between 28S rRNA and 18srRNA sequences. Genes of important enzymes in the mycotoxin biosynthetic pathway can be good targets for diagnostic tests as it will not only assure the presence of fungus but can also tell us about the mycotoxin production for the same gene which will add to specificity and sensitivity of the test (Baird R, Abbas H K, Windham G, Williams P, Baird S, Ma P, Kelley R, Hawkins L, Scruggs M. Identification of Select Fumonisin Forming Fusarium Species Using PCR Applications of the Polyketide Synthase Gene and its Relationship to Fumonisin Production in vitro. Int J Mol Sci. 2008 April;9(4):554-70, Atoui A, Mathieu F, Lebrihi A. Targeting a polyketide synthase gene for Aspergillus carbonarius quantification and ochratoxin A assessment in grapes using real-time PCR. Int J Food Microbiol. 2007 Apr. 20;115(3):313-8).
In the current invention a diagnostic assay for detection and identification of important Aspergillus species based on the gene of a key enzyme in polyketide biochemical pathway is developed, which will also add to specificity and sensitivity of the test. Potential mycotoxin production can be detected by PCR which may permit the establishment of critical control points and is a significant advantage. Aspergillus species are known to produce a wide range of secondary metabolites, under certain environmental conditions. Some of the important polyketides produced by Aspergillus species include Melanin pigments from A. fumigatus and carcinogenic mycotoxins Aflatoxins from A. flavus. Melanin is considered a virulent factor of Aspergillus fumigatus. Aspergillus flavus is also known to be an opportunistic pathogen of agricultural crops such as maize, cotton, groundnuts, rice, chillies and contaminate them with Aflatoxins and Sterigmatocystin. FAO approved permeable limits of Aflatoxin in agri products are 4-20 ppb in different countries (Jelinek C F, Pohland A E, Wood G E. Worldwide occurrence of mycotoxins in foods and feeds—an update. J Assoc Off Anal Chem. 1989 March-April;72(2):223-30). A. niger is also reported to produce ochratoxins, which contaminates nuts and coffee beans. Ochratoxin A, a polyketide product is teratogenic in rat, hamster and chick embryo and is an inhibitor of hepatic mitochondrial transport systems. It has also been reported to cause damage to the liver, gut, lymphoid tissue and renal tubular damage (Chulze S N, Magnoli C E, Dalcero A M. Occurrence of ochratoxin A in wine and ochratoxigenic mycoflora in grapes and dried vine fruits in South America.Int J Food Microbiol. 2006 Sep. 1; 111 Suppl 1:S5-9).
Diversity in end product produced by polyketide biosynthetic pathway by each Aspergillus species suggests the possible diversity in the structure and function of important enzymes such as Polyketide Synthase. Polykide synthase is key enzyme in the biochemical pathway responsible for production of polyketides and they are highly diverse in Aspergillus species. Polyketide Synthases of Aspergillus species are multidomain and multifuncational proteins of approximately 3000 amino acids with 7 to 9 domains encoded by a single gene (Schümann J, Hertweck C. Advances in cloning, functional analysis and heterologous expression of fungal polyketide synthase genes. J Biotechnol. 2006 Aug. 5; 124(4):690-703, Bhetariya P., Madan T, Varma A., Basir S, Sarma P U. Allergens/Antigens, Toxins and Polyketides of Important Aspergillus Species. Indian Journal of Clinical Biochemistry, June 2011. Vol. 26(2)104-119). The domains of the enzyme facilitate different steps in the synthesis of various intermediates of polyketide products. There are few domains essential for the minimal functional role of PKS while other domains are responsible for post polyketide modifications. Bioinformatics analysis of these Polyketide synthase domain sequences revealed conserved motifs and some non conserved sequences in the PKS domains. Based on the sequence analysis of these Polyketide synthases, sequences were identified and change in the sequences was identified. Information has been used to develop the diagnostic test for Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger in Agriculture samples for detection and identification of Aspergillus infections. Nucleotide sequences are selected and modified and probes have been developed for detection of Aspergillus species relevance to human and agriculture.
OBJECTIVES OF THE INVENTION
The objective of the present invention is to provide a PCR based simultaneous detection of Aspergillus species.
SUMMARY
Accordingly, the present invention provides a PCR based detection of Aspergillus species involving a conserved region of domain sequences from Polyketide synthase gene useful for detection and identification of important Aspergillus species. The diagnostic assay is based on identification of conserved region of domain sequences from Polyketide synthase gene useful for detection and identification of important Aspergillus species.



BRIEF DESCRIPTION OF FIGURES AND TABLES
Table 1: Sequence ID of primers and probes used in the study.
Table 2: Aspergillus Strains and isolates used in the study.
FIG. 1 (a): KS F-R primers gradient PCR with A. fumigatus,
FIG. 1 (b): KS F-R primers gradient PCR with A. flavus,
FIG. 1 (c): KS F-R primers gradient PCR with A. niger,
FIG. 2 (a): Specific amplification of A. fumigatus, A. flavus and A. niger using specific primers.
FIG. 2 (b): Gradient PCR for specific primers with (a) A. flavus (b) A. fumigatus and (c) A. niger.
FIG. 3. Multiplex PCR for A. fumigatus, A. flavus and A. niger.
FIG. 4 (a): Aspergillus fumigatus specific SYBR green Real time PCR.
FIG. 4 (b): Melting curve of Aspergillus fumigatus specific SYBR green Real time PCR.
FIG. 4 (c): Aspergillus fumigatus specific (probe Afu 5′ 6-FAM & 3′BHQ) Absolute amplification curve.
FIG. 5 (a): Aspergillus flavus specific SYBR green real time PCR.
FIG. 5 (b): Melting curve of Aspergillus flavus specific SYBR green real time PCR.
FIG. 5 (c): Aspergillus flavus specific (probe Afl 5′ HEX and 3′BHQ) Absolute amplification curve.
FIG. 6 (a): Aspergillus niger specific SYBR green Real time PCR.
FIG. 6 (b): Melting curve of Aspergillus niger specific SYBR green real time PCR.
FIG. 6 (c): Aspergillus niger specific (probe 5′ Ani TET and 3′ BHQ) Absolute amplification curve.
FIG. 7 (a): Aspergillus genus specific SYBR green Real time PCR.
FIG. 7 (b): Melting curve of Aspergillus genus specific (probe KS 5′ 6FAM and 3′BHQ).
FIG. 7 (c): Aspergillus genus specific (KS 5′ 6FAM and 3′BHQ) Absolute amplification curve.



DETAILED DESCRIPTION OF THE INVENTION
In the present work, PKS gene sequences of three important Aspergillus species A. fumigatus, A. flauvs and A. niger are aligned and analyzed. It was found that different species of Aspergillus have similar sequences or conserved region in the domains of PKS gene as well as some non-conserved region in domains of PKS gene. Sequence of PKS gene is represented by SEQ ID NO. 13. Specific primers and probes are developed based on the analysis for detection of A. fumigatus, A. flavus and A. niger together and specifically. Aspergillus fumigatus is a major contributing agent in systemic fungal infections and is often encountered in patients with organ transplants, acute leukemia. A. flavus and A. niger are also frequently reported in invasive cases in the recent past. Aspergillus flavus is an agricultural pathogen contributing to contaminations of carcinogenic Aflatoxins in groundnut, maize, cotton and tree nuts. A positive correlation between aflatoxin contamination of agricultural commodities and primary human hepatocellular carcinoma has been well documented (Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2009 February; 26(2):180-8). A. niger produced teratogenic mycotoxin ochratoxin A in coffee, nuts etc. A few immunologic tests exist for detection of these Aspergillus species with limited sensitivity and specificity. Polymerase chain reaction tests based on useful gene sequences in a ribosomal intergenic spacer region for Aspergillus species are reported which is genus specific and lacks species specificity (Rath P M, Ansorg R. Identification of medically important Aspergillus species by single strand conformational polymorphism (SSCP) of the PCR-amplified intergenic spacer region. Mycoses; 2000; 43(11-12):381-6). In recent efforts metabolic pathways are being examined for presence of fungi particularly on the mycotoxin producing potential of Aspergillus species. Probes are being designed based on mycotoxin biosynthetic pathway such as multiplex PCR is reported for Aflatoxin producers Aspergillus flavus, and Aspergillus parasaticus based on nor-1, ver-1, omt-A genes of aflatoxin (and sterigmatocystin) biosynthesis (Klingspor L, Loeffler J. Aspergillus PCR formidable challenges and progress. Med Mycol. 2009;47 Suppl 1:S241-7, Degola F, Berni E, Dall'Asta C, Spotti E, Marchelli R, Ferrero I, Restivo F M. A multiplex RT-PCR approach to detect aflatoxigenic strains of Aspergillus flavus. J Appl Microbiol. 2007 August;103(2):409-17.). It is well documented that there is a quantitative correlation between toxin production and biomass in naturally contaminated materials (Criseo G, Bagnara A, Bisignano G. Differentiation of aflatoxin-producing and non-producing strains of Aspergillus flavus group. Lett Appl Microbiol. 2001 October;33(4):291-5). Quantitative PCR systems do not use end point measurement to quantify the amount of target molecules present in the samples, while real time PCR systems detect the precise amount of target molecule present in the sample. This technology will be useful for determining associations between detection of a gene at critical control points in food production and quantification of the mycotoxin in the final product.
TaqManTM technology uses 5′-3′ exonuclease activity of polymerase to generate a template specific fluorescent signal after hydrolyzing an internal probe during each step of the PCR. The internal probe is 5′ labeled with a reporter fluorescent dye and 3′ ligated to a quencher dye; they are located in close proximity on the internal probe. The quencher dye greatly reduces the fluorescent emitted by the reporter dye by FRET. During PCR, the reporter dye is separated from the quencher Dye which results in an increase of the reporter dye signal. Only if the internal probe is binding to the DNA in between the two PCR primers a fluorescence signal during PCR is generated. Here an additional hybridization step increases the specificity of the PCR. Combining the quantitative detection and advantage of TaqManTM technology a sensitive and specific test can be developed which will indicate the presence of target molecule such as fungi itself and their potential to produce mycotoxin in the samples.
In the present invention the sequences identified from PKS domains are modified and fluorescent probes are prepared to use for identification of Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Probe for detection of three Aspergilli together such as Aspergillus fumigatus, Aspergillus flavus and Aspergillus nigeri based on conserved region in PKS domain is developed. Probe for Aspergillus fumigatus is specifically based on unique gene identified earlier (TMS33), Probe for detection of Aspergillus flavus is based on Polyketide synthase A protein (XP—002379951.1) and probe for detection of Aspergillus niger is based on Polyketide synthase protein (XP—001393884.2).
In an embodiment of the present invention oligonucleotide useful for the detection of Aspergillus species selected from the group consisting of SEQ ID NO.1-12.
In an embodiment of the present invention wherein SEQ ID NO. 1,2,4,5,7,8,10,11 are primers useful for PCR based detection of Aspergillus species.
In an embodiment of the present invention wherein SEQ ID NO. 3,6,9,12 are probes for PCR based detection of Aspergillus species.
In an embodiment of the present invention wherein the Aspergillus species is selected from the group comprising of A. fumigatus, A. flavus and A. niger. 
In another embodiment of the present invention provides a PCR based method for the detection of Aspergillus species comprising the steps of:

    
    
        a. isolating genomic DNA from the sample by known methods,
        b. designing primers having SEQ ID NO. 1, 2.
        c. performing Real time PCR using primers obtained in step b,
        d. optionally performing multiplex PCR using primers and probes having SEQ ID NO. 1,2,4,5,7,8,10,11.
        e. detecting and measuring the amount of amplified DNA.
    
    


In another embodiment of the present invention wherein the Aspergillus species is selected from the group comprising of A. fumigatus, A. flavus and A. niger. 
In another embodiment of the present invention the sequence detected is the polyketide synthase domain.
In another embodiment of the present invention the probe for detecting the nucleic acid sequence set forth as SEQ ID NO: 4,5,6 (forward primer, reverse primer and Taqman probe) from A. fumigatus specifically based on unique gene of A. fumigatus. 
In another embodiment of the present invention the probe for detecting the nucleic acid sequence set forth as SEQ ID NO: 7,8,9 (forward primer, reverse primer and Taqman probe) from A. flavus specifically.
In another embodiment of the present invention the probe for detecting the nucleic acid sequence set forth as SEQ ID NO: 10, 11, 12 (forward primer, reverse primer and Taqman probe) from A. niger specifically.
In another embodiment of the present invention wherein detecting the domain of Polyketide synthase gene nucleic acid sequence comprises use of a nucleic acid probe from the Aspergilli such as Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. 
In yet another embodiment of the present invention a kit for the diagnosis of Aspergillus species said kit comprising primers of SEQ ID NO. 1, 2, 4, 5, 7, 8, 10, 11 and probes of SEQ ID NO. 3, 6, 9, 12 optionally along with instructions manual.
In yet another embodiment of the present invention a kit for the diagnosis of Aspergillus species said kit comprising of:

    
    
        I. A primer set comprising a forward primer having SEQ ID NO. 1 and a reverse primer having SEQ ID NO. 2 wherein said forward primer and said reverse primer is capable of generating a PCR amplicon from a region of Polyketide synthase gene,
        II. A probe having SEQ ID NO. 3 capable of hybridizing to said PCR amplicon.
    
    


Based on genome sequences of Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger from NCBI, Polyketide synthase protein sequences are retrieved and aligned. The domains for PKS are searched by CDD search at NCBI and also by SEARCH PKs software available online. The sequences are then aligned by clustal X software. The conserved motifs in each domain are derived. One particular domain is selected which is highly conserved in these three fungi, the conserved motifs are derived by careful analysis of multiple alignment results. Up to 150 specific Amino acid sequences are taken and it is converted into nucleotide sequences by available universal amino acid code. Two degenerate primers are designed from these regions; degeneracy is taken care of by manually aligning the primer sequences. Primers are checked by BLAST. PCR conditions are optimized and primers are tested for specific amplification of product from A. fumigatus, A. flavus and A. niger. PCR is also tested for negative control such as Fusarium species. Around 60 samples are checked by this PCR from different isolates of A. fumigatus, A. flavus and A. niger. TaqManTM probe sequence is developed and checked for specific amplification.
From A. fumigatus a unique EST (TMS33, Gene ID: DN626065.1) is identified and reported (Upadhyay S, Shankar J, Madan T, Basir S, Sarma P U. Expressed sequence tags of Aspergillus fumigatus: extension of catalogue and their evaluation as putative drug targets and/or diagnostic markers. Indian Journal of Clinical Biochemistry, 2009/24 (2) 131-136). Gene sequence [Gene ID: 3507735 (AFUA—1G07280)] is taken from NCBI. It is hypothetical protein which does not have homology with any other fungi. Primers are designed and PCR conditions are optimized. DNA amplifications are performed using A. fumgiatus DNA. A product of 180 bp is amplified from A. fumigatus isolates.
Aflatoxin is a known carcinogenic mycotoxin for humans as well as animals. Aflatxins are synthesized by condensation of acetate units; their synthesis is estimated to involve at least 16 different enzymes. The enzymes and their gene sequences for aflatoxin biosynthetic pathway are now known. Polyketide SynthaseA (PKSA) is important in conservation of Acetate to Polyketide molecule, Norsolonic acid, one of stable intermediate of Aflatoxin Biosynthetic pathway. PKSA sequence from A. flavus and A. parasiticus are 98% homologous. PKSA protein (XP—002379951.1) from A. flavus is aligned with other PKS proteins of A. flavus, A. fumigatus and A. niger using with clustal X software. Non homologous region from PKSA is derived by careful analysis of the sequences. Selected amino acid sequences are translated into nucleotide sequences. Primers are designed and PCR conditions are optimized for amplification for specific product from A. flavus. PCR is also checked for non-specific amplification from A. fumigatus and A. niger. Amplification has been checked in toxigenic as well atoxigenic strains of Aspergillus flavus. TaqManTM probe sequence is developed and checked for specific amplification. A. flavus isolates from ITCC (Indian type culture collection) are collected, amplified and DNA is isolated.
PKSN (XP—001394705.1) from A. niger is aligned other PKS proteins of A. flavus, A. fumigatus and A. niger using with clustal X software. Non homologous region from PKSN is derived by careful analysis of the sequences. Selected amino acid sequences are translated into nucleotide sequences. Primers are designed and PCR conditions are optimized for specific amplification of DNA of A. niger. PCR is also checked for non-specific amplification from A. fumigatus and A. flavus. TaqManTM probe sequence is developed and checked for specific amplification of Aspergillus species.
The basic method for detection of A. fumigatus, A. flavus and A. niger is summarized as follows:

    
    
        1. Providing a template for the amplification of polyketide synthase gene from Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger for the detection. DNA is isolated from the clinical as well as agricultural samples as described in the protocol.
        2. Adding primers as shown in table 1 (SEQ ID NO. 1,2,4,5,7,8,10,11) for conducting multiplex PCR using the template obtained in step 1 for the amplification of polyketide synthase gene.
        3. Conditions used are 5 min initial step, followed by 35 cycles at 94° C. for 1 min, 56.7° C. for 1 min and 72° C. for 1.5 min and a final extension step at 72° C. for 5 min.
        4. Obtaining a detection of amplified product from A. fumigatus, A. flavus and A. niger by known method.
         Optionally method of detection of the A. fumigatus, A. flavus and A. niger is as follow.
        5. Providing a template for the amplification of polyketide synthase gene from
    
    


Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger for the detection. DNA template is isolated from the clinical as well as agricultural samples as described in the protocol.

    
    
        6. Adding primers and probes as shown in table 1 (SEQ ID NO. 1,2,3, 4,5,6,7,8,9,10,11,12) for conducting multiplex real time PCR using the template obtained in step 1.
        7. Conditions used are 50 cycles at 95° C. for 15 s and 60° C. for 1 min each, in ABI Real time PCR model 77005.
        8. Obtaining a detection of amplified product from A. fumigatus, A. flavus and A. niger by known method.
    
    


Details of sequences of polyketide synthase gene and proteins from various Aspergillus species are as follows:
Gene Sequences







GCTTGAGGTCTCCTGTAAGGAAGCCCATCATAATTTCTTGATTGAAAGCG


 


ATTTGGTCCAGTTGATTAACCATTGTGCCTTGTCTAGACGGTAAGATGAA


 


GCAAACATCCGAGGAGTATGTTCCCGGGGAAGCTACAGAGGAGGTAACTG


 


CAGAAGCAAGTGCAACCTCTGATCCTACGTTGGCGAATGCGGCCTACACT


 


GAACTCCAGGACGGCCCTCTTGACGCCGGTGCTACAGCAGCCAACGAGAT


 


TGACTCCTCTGCGCCTAAAGCTGAGGTCTCTCCACCCGCGCAGACTCTCG


 


TTTCCGATGCTGCTAATCCTGTAGCCGAGGCGTCGTGGGAACAGAACGGG


 


GCTGGCTCGCTCGAGTCGTCGGCAAATGCTGACGGCTGGGTTGAGGTGCC


 


TCGCGACCCGGCGGAGACGGAGACGGGCCTACAGGCCACCCCTGCCTCTG


 


TCGATACCGGTCTGAAGGACAACCAGACTGTCGCTGCGGCCTCCGGACAG


 


AGTGATGAACATGTCTCCGTGCCTAAGGCGCAGGGCAGCGACGGATTCGA


 


ACCCGTTGTG






Protein Sequences









SEQ ID No. 14:



ref|XP_002379951.1| aflC/pksA/pksL1/polyketide synthase [Aspergillus


flavus NRRL3357] (311-394)


>tlleqvrldl vetglprllq srqvksvtiv pfltrmnetm snilpvsfis tetrtdtgra ipasgrpgag kcklaivsms


 


grfpesptte sfwdllykgl dvckevprrr wdinthvdps gkarnkgatk wgcwldfsge fdprffgisp keapqmdpaq


 


rmalmstyea meraglvpdt


 


SEQ ID No. 15:


ref|XP_001398521.2| polyketide synthase [Aspergillus niger CBS 513.88]


(1956-2132)


>pdktyllagglgglgrtlaewmlqrnakhlvflsrsgetraeakatvswlrahgidvtvykgdvanpadvqacvggirnlggvfha


 


amvladaalenmtyaqwhqcvqpkvvgafnlhqatkslpldffvtfssvsacfgtrsqgnyaaantyldalmryrrqiglpaatmn


 


cgrit


 


SEQ ID No. 16:


gi|71002828|ref|XP_756095.1| conidial pigment polyketide synthase


PksP/Alb1 [Aspergillus fumigatus Af293]


> qdidtyfipg gnraftpgri nyyfkfsgpsvsvdtacsss laaihlacna iwrndcdtai sggvnlltnp dnhagldrgh


 


flsrtgncntfddgadgycr adgvgtivlk rledaeadnd pilgvinaay tnhsaeavsi trphvgaqafifnkllndtn


 


tnpheigyve mhgtgtqagd avemqsvldv fapdyrrgpa nslylgsaksnighgesasg vtslvkvllm lkqnmipphc


 


giktkinhnf ptdlaqrnvh iafkptpwnr


 


SEQ ID No. 17:


gi|238503167|ref|XP_002382817.1| polyketide synthetase PksP [Aspergillus


flavus NRRL3357]


>tsddyreins gqdidtyfip ggnraftpgr inyyfkfsgpsvsvdtacss slaaihmacn siwrndcdaa iaggvniltn


 


pdnhagldrg hflsrtgncntfddgadgyc radgvgtiil krledaqadn dpilgvinga ytnhsaeays itrphvgaqa


 


fifnkllnda nidpkdvsyv emhgtgtqag davemqsvld tfapdyrrgp gqslhlgsakanvghgesas gvtalvkvll


 


mmkkntipph cgiktkinhn fptdlaqrnv hiafqptpwn


 


SEQ ID No. 18:


gi|317031606|ref|XP_001393884.2| conidial yellow pigment biosynthesis


polyketide synthase [Aspergillus niger CBS 513.88]


>nraftpgrin yyfkfsgpsv svdtacsssl aaihmacnsiwrndcdaait ggvniltspd nhagldrghf lsttgncntf


 


ddgadgycra dgvgsivlkrledaeadndp ilavingayt nhsaeavsit rphvgaqafi fnkllndani


 


dpkdvsyvemhgtgtqagda vemqsvldvf apdyrrgpgq slhigsakan ighgesasgv talvkvllmm


 


renmipphcg iktkinsnfp tdlakrnvhi afqptpwnrp asgkrrtfvn nfsaaggnta


 


llledapipe rqgqdprsfh






EXAMPLES
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention
Examples 1
Isolation of Fungal DNA
Aspergillus isolates are grown on SDA Broth (Sabouraud Dextrose Broth) (Himedia) for 3-5 days at 37° C. degree to generate fungal growth for DNA extraction.
Fungal biomass/mycelia are harvested after 3 days, filtered and washed several times with sterile distilled water. Mycelial mat is transferred into a pre-cooled (−20.degree C.) sterile ceramic mortar, flushed with liquid nitrogen, and slowly ground with a pestle into a fine powder. Four grams wet weight of mycelia paste is collected in a 15 ml polypropylene screw cap tube (Falcons, Germany). Fungal cells are suspended in 4 ml of 10mM phosphate buffer, pH 6.0 and incubated with 0.5 U chitinase per gram wet weight of cells at 25° C. for 90 mins At the end of the incubation period, equal volume of lysis buffer (50 mM Tris-cl, 50 mM EDTA, 3% SDS, 1% β-mercaptoethanol pH 7.2) is added and the suspension is incubated at 65° C. in a water bath for 1 hour. Subsequently, the contents of the polypropylene tube are subjected to heat treatment in a microwave oven for 3 sec each for three times.
The cell lysate is extracted with phenol:chloroform:isoamylalcohol (25:24:1) and the DNA in the aqueous layer is precipitated with two volumens of ethyl alcohol in the presence of 0.1M sodium acetate. DNA is washed with 70% ethanol, dried and dissolved in TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8). The DNA sample is then treated with RNase A (50 μg/ml) for 30 min at 37° C. The sample is extracted with phenol:chloform:isoamylalcohol and precipitated with ethanol as above. The DNA is dissolved in TE buffer and the concentration is determined spectrophotometrically. Quality of the DNA is also checked by gel electrophoresis. The powder is suspended in Lysis Buffer (Containing Tris, EDTA, RNAse) containing RNase (Sigma Chemical Co., St. Louis, Mo.), and transferred into an Oak Ridge centrifugation tube (Nalge Nunc International, Rochester, N.Y.). A rapid method for extraction of genomic DNA based on the cleavage of chitin with chitinase is developed in house (Bir N, Paliwal A, Muralidhar K, Reddy P, Sarma P U. A rapid method for the isolation of genomic DNA from Aspergillus fumigatus. Prep Biochem. 1995 November; 25(4):171-81).
Extraction of Aspergillus DNA from Biological Samples
The sputa and bronchial aspirates are incubated with 1% pancreatin at room temperature for 1 hour. The fluidized samples are centrifuged at 3500×g for 20 mins. Pellets are washes with 1 ml of 0.1 M phosphate buffer and recentrifuged. Swabs are taken from the pellets and investigated for the presence of Aspergillus fumigatus by culture. The pellet is suspended in 0.2 ml of 0.1 M phosphate buffer, pH 6.8. Control specimens from healthy individuals are also processed under similar conditions.
Aliquots from suspended pellets are taken in eppendrof tubes and 0.5 U of chitinase is added to each tube and incubated for 1 hour at 25° C. tubes are then heated in a microwave oven for 3 s cycles each. Debris from the chitinase treated sputa and bronchial aspirate is pelleted by centrifugation at 12,000×g for 10 min The supernatant is siphoned off and the pellet is emulsified by vigorous vortexing in 100 μl if chloroform. Distilled water (50 μl) is added and emulsified with the chloroform phase. After centrifugation at 12,000×g for 10 min, the upper aqueous phase is collected. The samples containing the chromosomal DNA are stored at −20° C. with choloroform, and are recentrifuged prior to use.
Extraction of DNA from Agricultural Samples.
Two grams of agricultural produce (e.g. ground nut seeds) is resuspended in 10 ml of SDA broth and incubated in 50-ml sterile Falcon tubes on an orbital shaker with gentle agitation (50 rpm) at 37° C. for 48 hours. After incubation, the tube contents are centrifuged (5 min at 5,000 3 g) and the pellets are frozen in liquid nitrogen. From suspension blends, 2-ml portions are taken and total DNA is extracted by the above-described procedure.
Preparation of Primers and Probes
All primers and probes are synthesized by automated DNA synthesizer (Lab India, ABI Prism).







TABLE 1







Sequence ID of primers and probes used in the study.















Amplifi-


Sequence
Sequence


cation


ID no.
ID/name
Primer/probe sequence
Species
product





 1
KS-F
ATCTGGAGAAATGAYTGCGATGCYGCY
A.
200 bp




AT*
fumigatus,






A. flavus






and A.






niger



 


 2
KS-R
TCKAGACCGGYATGGTTYTC*
A.






fumigatus,






A. flavus






and A.






niger



 


 3
KS-
6-FAM
A.




probe
5′AACCAYGCCGGTCTKGAYCGBGGCC
fumigatus,





A3′ BHQ1*
A. flavus






and A.






niger



 


 4
Afu F
TAAGATGAAGCAAACATCCGAGGAGT
A.
180 bp





fumigatus



 


 5
Afu R
GCGGGTGGAGAGACCTCAGCT
A.






fumigatus



 


 6
AfuProbe
6-
A.





FAM5′GTGCAACCTCTGATCCTACGTTG
fumigatus





GC3′BHQ1




 


 7
AflF
CGATTGATCACAAGTTGGCTCGAAC
A. flavus
250 bp


 


 8
AflR
TACATGTTGCCAGATTCCTCATATTCCC
′′





TAG




 


 9
Aflprobe
HEX-5′
′′





GCGCCAAATGGTCCAGAAGTATGTC






3′BHQ1




 


10
AniF
CAACGCAAAA′TATGGCTACT′ATCTCG
A. niger
110 bp




ATCA




 


11
AniR
CATTGATTTCTTCCAGGGTGATTCCG
′′



 


12
AniProbe
TET-5′
′′





GAAGCTTCTTCCACATCTCAGGCAAGG






A3′BHQ1





*R = AG, Y = CT, M = AC, K = GT, W = AT, S = CG, B = CGT, D = AGT, H = ACT, V = ACG, N = ACGT






1) A conserved region of Polyketide synthase domain is identified by multiple alignments of Polyketide synthases gene from A. fumigatus (XP—756095.1), A. flavus (XP—002382817.1) and A. niger (XP—001398521.2). Primers are designed and PCR conditions are optimized (table 1). DNA amplifications are performed A product of 200 bp is amplified from A. fumigatus, A. flavus and A. niger (FIG. 1a, 1b and 1c), and table 2). The TaqmanTM probe specific for A. fumigatus, A. flavus and A. niger is designed (SEQ ID NO. 1,2,3) by following the general rules outlined by the manufacturer using Primer Express software, version 2.0 and are synthesized at Biochem ltd. A probe detecting A. fumigatus, A. flavus and A. niger specific 200 bp amplicon contained the reporter dye FAM covalently attached to the 5′ end and the quencher BHQ at 3′ TaqmanTM assay is carried out in 50-μl volume reactions, with the additions 12.5 μl of TaqMan Universal Master Mix, 2.5 μl of forward and reverse primers (10 nM each), 2.5 μl of TaqMan probe, 2.5 μl of 2 mg/ml bovine serum albumin, fraction V and 5 μl of DNA template. Standard procedures for the operation of the model 7700 as described in the PerkinElmer instrument's manual, are followed. The temperature cycling (50 cycles at 95° C. for 15 s and 60° C. for 1 min each) is performed in a 96-well thermal cycler. Each amplification run contained several negative controls. Amplification data collected by the 7700 Sequence Detector are then analyzed by the use of the Sequence Detection System software. The fractional cycle number reflecting a positive PCR result is called the cycle threshold (Ct). Control sample without DNA template is included in the experiment runs as negative control. All samples except the controls are tested in duplicate; the control is tested in triplicate (FIG. 7a, 7b and 7c, table 2).







TABLE 2







Aspergillus Strains and isolates used in the study
















Multiplex
Real time


S. No.
Strains
ATCC NO.
Source
PCR
PCR results












Strains used for Primers and probe design












1
Aspergillus fumigatus
Aspergillus
Patient lung
−
−




fumigatus
suffering from




Af293
IA


2
Aspergillus flavus
Aspergillus
Cotton seed
−
−




flavus




NRRL3357


3
Aspergillus niger
Aspergillus
—
−
−




niger CBS




513.88









Strains used for PCR and Real time PCR












1.
Aspergillus flavus
NRRL18079
Cotton seeds
+
+


2.
Aspergillus flavus
NRRL2211
—
+
+


3.
Aspergillus flavus
MTCC 1884
Vegetable
−
+





waste


4.
Aspergillus fumigatus
ATCC13073

+
+


5.
Aspergillius fumigatus
ITCC2550
Uranium
+
+




(MTCC no)
waste


6.
Aspergillius fumigatus
MTCC No.
Mangroove
+
+




7132
soil


7.
Aspergillus niger
ITCC2218.95
Apple orchard
−
+


8.
Aspergillus niger
ATCC9029
—
+
+


9.
Aspergillius niger
ATCC6275
—
+
+









2) A unique EST (TMS33, Gene ID: DN626065.1) from A. fumigatus is identified and reported (28). Gene sequence (Gene ID: 3507735 (AFUA—1G07280) is taken from NCBI. Primers are designed and PCR conditions are optimized. DNA amplifications are performed. A product of 180 bp is amplified from A. fumigatus (FIG. 2a, 2b). The TaqmanTM probe specific for A. fumigatus is designed (SEQ ID NO. 4,5,6) by following the general rules outlined by the manufacturer using Primer Express software, version 2.0 and are synthesized at Biochem Ltd. A probe detecting A. fumigatus specific 180 bp amplicon contained the reporter dye FAM covalently attached to the 5′ end and the quencher BHQ attached to the 3′. The TaqmanTM assay is carried out in 50-μl volume reactions with the additions 12.5 μl A of TaqMan Universal Master Mix, 2.5 μl of forward and reverse primers (10 nM each), 2.5 μl of TaqMan probe, 2.5 μl of 2 mg/ml bovine serum albumin, fraction V and 5 μl of DNA template. Standard procedures for the operation of the model 7700 as described in the perkin almer instrument's manual, are followed. The temperature cycling (50 cycles at 95° C. for 15 s and 59° C. for 1 min each) is performed in a 96-well thermal cycler. Each amplification run contained several negative controls. Amplification data collected by the 7700 Sequence Detector are then analyzed by the use of the Sequence Detection System software. The fractional cycle number reflecting a positive PCR result is called the cycle threshold (Ct). Control sample without DNA template is included in the experiment runs as negative control. All samples except the controls are tested in duplicate; the control is tested in triplicate (FIG. 4a, 4b, 4c, table 2).
3) A region of Polyketide synthase A protein sequence from A. flavus (accession no.:—XP—002379951.1) is taken from NCBI. Selected AA sequences are converted to nucleotide sequences by DNASTAR software. Primers are designed and PCR conditions are optimized. DNA amplifications are performed. A product of 250 bp is amplified from A. flavus (FIG. 2a, 2b). The TaqmanTM probe specific for A. flavus is designed (SEQ ID NO. 7,8,9) by following the general rules outlined by the manufacturer using Primer Express software, version 2.0 and are synthesized at Lab India. A probe detecting A. flavus specific 250 bp amplicon contained the reporter dye HEX covalently attached to the 5′ end and the quencher BHQ at 3′. The TaqmanTM assay is carried out in 50-μl volume reactions with the additions 12.5 μl of TaqMan Universal Master Mix, 2.5 μl of forward and reverse primers (10 nM each), 2.5 μl of TaqMan probe, 2.5 μl of 2 mg/ml bovine serum albumin, fraction V and 5 μl of DNA template. Standard procedures for the operation of the model 7700 as described in the perkin almer instrument's manual, are followed. The temperature cycling (50 cycles at 95° C. for 15 s and 59° C. for 1 min each) is performed in a 96-well thermal cycler. Each amplification run contained several negative controls. Amplification data collected by the 7700 Sequence Detector are then analyzed by the use of the Sequence Detection System software. The fractional cycle number reflecting a positive PCR result is called the cycle threshold (Ct). Control sample without DNA template is included in the experiment runs as negative control. All samples except the controls are tested in duplicate; the control is tested in triplicate (FIG. 5a, 5b, table 2).
4) A region of Polyketide synthase protein sequence from A. niger (accession no.:—XP—001398521.2) is taken from NCBI. Selected AA sequences are converted to nucleotides.
Primers are designed and PCR conditions are optimized. DNA amplifications are performed. A product of 110 bp is amplified from A. niger (FIG. 2a, 2b). The TaqmanTM probe specific for A. niger is designed (SEQ ID NO. 10,11,12) by following the general rules outlined by the manufacturer using Primer Express software, version 2.0 and are synthesized at Lab India. A probe detecting A. niger specific 110 bp amplicon contained the reporter dye TET covalently attached to the 5′ end and the quencher BHQ attached to the 3′. The TaqmanTM assay is carried out in 50-μl volume reactions with the additions 12.5 μl of TaqMan Universal Master Mix, 2.5 μl of forward and reverse primers (10 nM each), 2.5 μl of TaqMan probe, 2.5 μl of 2 mg/ml bovine serum albumin, fraction V and 5 μl of DNA template. Standard procedures for the operation of the model 7700 as described in the perkin almer instrument's manual, are followed. The temperature cycling (50 cycles at 95° C. for 15 s and 59° C. for 1 min each) is performed in a 96-well thermal cycler. Each amplification run contained several negative controls. Amplification data collected by the 7700 Sequence Detector are then analyzed by the use of the Sequence Detection System software. The fractional cycle number reflecting a positive PCR result is called the cycle threshold (Ct). Control sample without DNA template is included in the experiment runs as negative control. All samples except the controls are tested in duplicate; the control is tested in triplicate (FIG. 6a, 6b).
Quantification of Real Time PCR
The fractional cycle number reflecting a positive PCR result is called the cycle threshold (Ct). Control sample without DNA template is included in the experiment runs as negative control. Absolute Quantification of Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger by CT method as determined from TaqMan analysis.
Quantification is performed by first subtracting mean reference sequence CT values from target sequence CT values for both test samples and a pre specified calibrator sample to obtain Delta CT values.
ADVANTAGES OF THE INVENTION


    
    
        1. The PCR based simultaneous detection of A. fumigatus, A. flavus and A. niger from any biological samples is very useful diagnostic method.
        2. The method can indicate the toxigenic potential of the organism based on the gene amplification.
        3. The method is economical compared to other available immunoassays.
        4. The method is faster and can be applicable for screening of large number of samples.

## Claims

1. A kit for the detection of Aspergillus species, said kit comprising as primers the oligonucleotides of SEQ ID NO. 1, 2, 4, 5, 7, 8, 10 and 11, and as probes the oligonucleotides SEQ ID NOS: 3, 6, 9 and 12, wherein each probe contains a fluorescent label, with instructions for use of said oligonucleotides for detection of Aspergillus species.

2. A set of oligonucleotides useful for the detection of Aspergillus species containing oligonucleotides SEQ ID NOS: 1, 2, 4, 5, 7, 8, 10 and 11 as primers for PCR-based detection of Aspergillus species and oligonucleotides SEQ ID NOS: 3, 6, 9, and 12 as probes for PCR-based detection of Aspergillus species,wherein each probe contains a fluorescent label.

3. A PCR-based method for the detection of Aspergillus species comprising the steps of:
a. providing DNA from a sample and
b. performing multiplex PCR on said DNA using as primers the oligonucleotides SEQ ID NOS: 1, 2, 4, 5, 7, 8, 10 and 11, and
c. detecting the PCR amplification products.

4. The method of claim 3 wherein the PCR amplification products are detected using as probes the oligonucleotides SEQ ID NOS: 3, 6, 9 and 12, wherein each probe contains a fluorescent label.