Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-03-27
2003-09-23
Whisenant, Ethan (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300, C536S024320, C536S023700, C435S091100
Reexamination Certificate
active
06623932
ABSTRACT:
FIELD OF THE PRESENT INVENTION
The present invention relates to three sets of novel primers of SEQ ID Nos. 1-6, wherein said three sets of primer are designed from three genes omt, ord, and afl R respectively of aflatoxin biosynthesis pathway of fungi
Aspergillus flavus
and an improved method of identifying aflatoxinogenic aspergilli using said three sets of primers.
BACKGROUND OF THE PRESENT INVENTION
Aflatoxins are potent carcinogenic, mutagenic and teratogenic metabolites produced primarily by the fungal species of
Aspergillus flavus
and
Aspergillus parasiticus.
Foods and feeds, especially in warm climates are susceptible to invasion by aflatoxigenic Aspergillus sp. And subsequent production of Aflatoxins during preharvesting, processing, transportation or storage. Over the last few years, means for mycotoxin detection have been simplified, by the adoption of immunological methods. The level of mold infestation and identification of the governing species are important parameters which could give an indication of the quality of the material and future potential for the presence of mycotoxins.
Mold counts are a part of quality control assurance for foods. This method is time consuming, labour intensive, costly requires facilities and mycological expertise and do not allow the specification of mycotoxegenic fungi.
With the advances made in the detection methods, polymerase chain reaction (PCR) facilitates in vitro amplification of target sequence and offers several advantages over traditional methods of detection.
Reference may be made to the work of Miller and Martin (1988) for the application of PCR techniques for the detection of microorganisms, including plant pathogens. However no attempt has been made to detect aflatoxin-producing fungi.
Reference may be made to the works of Payne and Woloshuk (1989) and Nu, et al (1995). Who have identified the genes in Aflatoxins biosynthetic pathway of strains of
A. flavus
and
A. parasiticus.
However, detection of aflatixigenic fungi were not attempted.
Reference may be made to the work of Shapira, et al (1996), where in the identification of aflatoxin producing molds in grains has been attempted using PCR techniques. Three genes ver-1, omt-1 and apa-2 coding for key enzymes and regulatory factor in biosynthesis of aflatoxin were used as primers. Positive results were obtained in 24 h enriched cultures at lowest spore level of 10
2
spores per gram. However incubation of dried ground corn seeds in enrichment media allowed detection as few as 10
2
spores per gram after 48 h of incubation.
The drawback of these references is that no attempts have been made to detect aflatoxigenic fungi and the traditional methods are non-sensitive, time consuming and lack consistency. The present invention enables detection of aflatoxigenic fungi by PCR using specific aflatoxin biosynthetic pathway genes. This method has application in food system.
OBJECTS OF THE PRESENT INVENTION
The main object of the present invention is to develop primers from genes of aflatoxin biosynthesis pathway.
Another main object of the present invention is to develop primers for genes omt, ord, and afl R of aflatoxin biosynthesis pathway.
Yet another object of the present invention is to develop an improved method for identifying aflatoxinogenic aspergilli.
Still another object of the present invention is to develop an improved method for identifying aflatoxinogenic aspergilli using said primers.
Still another object of the present invention is to develop a method of identifying aflatoxinogenic aspergilli directly in food articles.
Still another object of the present invention is to develop a highly sensitive method to identifying aflatoxinogenic aspergilli.
Further object of the present invention is to develop an identification method using multiple number of primers for more accuracy.
SUMMARY OF THE PRESENT INVENTION
The present invention relates to three sets of novel primers of SEQ ID Nos. 1-6, wherein said three sets of primer are designed from three genes omt, ord, and afl R respectively of aflatoxin biosynthesis pathway of fungi
Aspergillus flavus
and an improved method of identifying aflatoxinogenic aspergilli using said three sets of primers.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
Accordingly, the present invention relates to three sets of novel primers of SEQ ID Nos. 1-6, wherein said three sets of primer are designed from three genes omt, ord, and afl R respectively of aflatoxin biosynthesis pathway of fungi
Aspergillus flavus
and an improved method of identifying aflatoxinogenic aspergilli using said three sets of primers.
In one embodiment of the present invention, three sets of oligonucleotide primers of SEQ ID Nos. 1 through 6. (Please refer sequences shown below).
omt 1 (F) 5′ AGCGTCCGAATCCCTTTAAT 3′ (SEQ ID NO. 1)
(R) 5′ AGGGTGTTCGCCAATCATAG 3′ (SEQ ID NO. 2)
ord (F) 5′ ACTGCCCCTCAGCTAACCTC 3′ (SEQ ID NO. 3)
(R) 5′ GCATCAGCATTCTTCCAAGG 3′ (SEQ ID NO. 4)
aflR (F) 5′ AACCGCATCCACA ATCTCAT 3′ (SEQ ID NO. 5)
(R) 5′ AGTGCAGTTCGCTCAGAACA3′ (SEQ ID NO. 6)
In further embodiment of the present invention, said three sets of primer of SEQ ID Nos. 1-6 used together shows more accurate identification as compared to any one set of primer alone.
In another embodiment of the present invention, wherein said primers are designed from genes of aflatoxin biosynthesis pathway of fungi
Aspergillus flavus.
In yet another embodiment of the present invention, wherein said primers are designed for three specific genes omt, ord, and afl R.
In still another embodiment of the present invention, wherein primers 1 and 2 correspond to gene omt encoding o-methyl transferase.
In still another embodiment of the present invention, wherein primers 3 and 4 correspond to gene ord encoding oxidoreductase.
In still another embodiment of the present invention, wherein primers 5 and 6 correspond to gene afl R encoding aflatoxin regulatory protein.
In still another embodiment of the present invention, wherein primers 1, 3, and 5 are forward primers.
In still another embodiment of the present invention, wherein primers 2, 4, and 6 are reverse primers.
In still another embodiment of the present invention, wherein length of primers is 20 base pairs (bp).
In further embodiment of the present invention, sequences of three genes omt, ordA, and afl R are as follows:
A. O methyltransferase (Omt) gene from the published gene sequence with Accession no L25834 where the primers covers the region between 1811 to 2218 with the product size 407 bp of SEQ ID NO. 7.
B. Oxidoreductase (ord) gene from the published gene sequence with Accession no AF 169016 where the primer covers the region between 3142 to 3530 with product size 388 bp of SEQ ID NO. 8.
C. Aflotoxin regulatory gene (aflR) from the published gene sequence with Accession no AF 264763 where the said primer covers the region between 540 to 1338 with the product size 798 bp of SEQ ID NO. 9.
In further embodiment of the present invention, an improved method of identifying aflatoxigenic aspergilli using primers of claim
1
independently or in combination.
In another embodiment of the present invention, harvesting mixed microflora from food system.
In yet another embodiment of the present invention, extracting DNA from said harvested flora. (Please refer
FIGS. 1
, and
2
)
REFERENCES:
patent: 6372430 (2002-04-01), Morrison et al.
Yu,J. et al. Comparison of the omtA genes encoding O-methyltransferases involved in aflatoxin biosynthesis fromAspergillus parasiticusandA. flavus. Gene 163 (1), 121-125 (1995).*
Yu,J. et al. Cloning and characterization of a cDNA fromAspergillus parasiticusencoding an O-methyltransferase involved in aflatoxin biosynthesis. Appl. Environ. Microbiol. 59 (11), 3564-3571 (1993).*
Yu, J. et al Genes encoding cytochrome P450 and monooxygenase enzymes define one end of the aflatoxin pathway gene cluster inAspergillus parasiticus. Appl. Microbiol. Biotechnol. 53 (5), 583-590 (2000).*
Yu, J. et al Characterization of the critical amino acids of
Chandrashekar Arun
Manonmani Haravey Krishnan
Rao Eddiya Rati
Council of Scientific and Industrial Research
Hashemi Shar
Mathews, Collins, Shepherd & McKay. P.A.
Whisenant Ethan
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