detection of mycotoxigenic fungi and characterization of...
TRANSCRIPT
Application of molecular biological techniques for detection of mycotoxigenic fungi and
characterization of genes responsible for toxin production
Ph. D. Thesis Submitted by
Ahmed A. El Hamaky
Under supervision of
Prof. Dr. Heidy M. Shawky
Prof. Dr. Mohamed K. Refai
Prof. Dr. Atef A. Hassan
Cairo University, 2016
Introduction
Nearly every food or feed commodity can be contaminated by fungal pathogens and many of these fungi are capable of producing one or more mycotoxins.
Because of the toxic and carcinogenic potential of produced mycotoxins , there is an urgent need to develop recent detection methods that are rapid and highly specific.
MYCOTOXINS
•Toxic chemical products produced by
fungi.
•The fungus consume organic matter
whenever humidity and temperature are
sufficient for their growth.
•The actual reason for the production of
mycotoxins is not yet known; they are
neither necessary for fungal growth nor the
development of the fungi.
Major mycotoxins
Aspergillus toxins: the most important are
Aflatoxins are mycotoxins produced by Aspergillus species of fungi, such as A. flavus and A. parasiticus
Ochratoxin is a mycotoxin produced by Penicillium and Aspergillus species.
Penicillium toxins as citrinin, patulin
Fusarium toxins asfumonisins, zearalenone
Other toxins as amatoxins and phallotoxins
Detection of mycotoxins
1. Detection and estimation of mycotoxins:
Analytical methods ( TLC, HPLC & MS ). Immunological methods (Radio Immuno Assay
(RIA), Enzyme Immuno Assay (EIA) or Enzyme-Linked Immuno Sorbent Assay (ELISA)
2. Identification of mycotoxigenic molds by
molecular biological techniques: which are more rapid and more objective methods for
the identification of mycotoxigenic fungi in human foods and animal feeds
Aflatoxins
They are a types of mycotoxins
produced by Aspergillus species of fungi,
such as A. flavus and A. parasiticus .
Among various mycotoxins, aflatoxins have assumed higher significance due to their carcinogenic effects on human beings, poultry and livestock (domestic animals).
Natural occurrence of AFs
Agricultural products contaminated with aflatoxins include:
1. Cereals (maize, rice, wheat).
2. Oil seeds (groundnut, soybean, sunflower, cotton).
3. Spices (black pepper, coriander, turmeric, zinger).
4. Tree nuts (almonds, pistachio, walnuts, coconut).
5. Milk.
Properties
18 different types of aflatoxins were identified. The major important members are aflatoxin B1,
B2, G1 and G2. Based on their fluorescent color when exposed
to ultraviolet light.(B series = blue fluorescence) (G series = yellow-green fluorescence). The aflatoxins fluoresce strongly in UV (365 nm). Aflatoxin M1 and M2 are major metabolites of
aflatoxin B1 and B2 respectively, found in milk of animals that have consumed feed contaminated with aflatoxins B1 and B2 .
Properties (Cont.)
Aflatoxins M1, M2 may be found in milk and other aflatoxins may be absent.
Aflatoxin B1 (AFB1) is normally predominant in cultures as well as in food products (the most dangerous type of Afs).
Aflatoxins are slightly soluble in water.
Soluble in moderately polar organic solvents.
Insoluble in non polar solvents.
Aflatoxins decompose at their melting points, which are between 237°C (G1) and 299°C (M1),
Not destroyed under normal cooking conditions.
Chemical Structure of AFs
They are classified into two broad groups:
1- Difurocoumarocyclopentenone Series.
(B series) (AFB1 , AFB2 , AFB2A ,
AFBM1 , AFBM2, AFBM2A and
aflatoxicol).
AFB1
Structure (Cont.)
2- Difurocoumarolactone series .
(G series) (AFG1 , AFG2 , AFG2A , AFGM1
AFGM2 , AFGM2A).
AFG1
O O
O
O
O
O
OCH3
Toxicity of AFs
The following effects may occur singly or in combination:
1-Aflatoxins have carcinogenic effect in human and animal ( cause hepatocellular carcinoma )
2- Teratogenic
3- Mutagenic
4- Immunosuppressive.
Carcinogenicity(Most danger effects)
Aflatoxins are known to be human
carcinogens (hepatocellular carcinoma, or
primary liver-cell cancer).
The risk of liver cancer increased significantly
with increasing prolonged exposure to AFs
consumption.
Biomarkers for aflatoxin exposure (aflatoxin
metabolites in the urine and aflatoxin albumin
adducts in the blood)
Preventing exposure to aflatoxin
The traditional approach to preventing exposure to aflatoxin via consumption of feed or food .
Feed additive of antioxidants as zinc and selenium , vitamins and sorbents as silica and clay to ameliorate the toxic effects of Afs which may be present in feed
.
Preventing exposure to aflatoxin (Cont.)
Chemoprotection is based on manipulating the biochemical processing of aflatoxin to ensure detoxification rather than preventing biological exposure.
Enterosorption is based on the approach of adding a binding agent to food to prevent the absorption of the toxin while the food is in the digestive tract; the combined toxin-sorbent is then excreted in the feces.
Recently, feed additive of nanoparticles of Zinc and Selenium were successfully used (Hassan et al., 2016)
Ochratoxin
Ochratoxins are a group of mycotoxins produced as secondary metabolites by several fungi of the Aspergillus and Penicillium spp.
They are weak organic acids consisting of isocoumarin derivatives
coupled to phenylalanine.
Properties of OA
The family of ochratoxins consists of three members, A, B, and C.
Ochratoxin A is the most abundant , commonly detected in feed and the most toxic one.
It is a potent toxin affecting mainly the kidney.
O A can contaminate a wide variety of foods and feed as a result of fungal infection in the field during growth, at harvest, in storage and in shipment.
Properties (Cont.)
Aspergillus ochraceus is the most known species of ochratoxin –producing Aspergillus.
Aspergillus niger is also found to be a producer of ochratoxin
They grow at moderate temperatures and at a high water activity and is a significant source of ochratoxin A in cereals.
Natural occurrence of OA
Ochratoxin A is found mainly in cereal and cereal products.
In coffee, spices, dried fruits, grape juice.
Meat and meat products of non-ruminant animals (chickens, rabbits) exposed to feedstuffs contaminated with this mycotoxin.
Toxicity and health implications
Ochratoxin A is a toxic carcinogenic fungal toxin found in a variety of food.
Ochratoxin A is absorbed from the gastrointestinal tract.
The absorbed ochratoxin A is distributed via blood, mainly to the kidneys, and at lower concentrations to the liver, muscle and fat.
Toxicity
The subchronic and chronic effects of ochratoxin A are of greatest concern. Ochratoxin A has been shown to be nephrotoxic, hepatotoxic, teratogenic and immunotoxic to several species of animals and carcinogenic in mice and rats causing tumors of the kidney and liver.
The acute toxicity of ochratoxin A is relatively low.
At present, there are no documented cases of acute toxicity reported in humans.
The plan of the present study
Isolation and Identification of mycotoxigenic molds from feed and feedstuffs by traditional methods
Evaluation of isolated molds for mycotoxin production on synthetic medium (yeast extract) and yellow corn.
Establishing a PCR protocols by use of universal fungal primers for the detection and differentiation of a broad spectrum of fungal DNA.
Detection of mycotoxigenic fungi using PCR technique to detect genes implicated in the mycotoxin production cycle.
Samples
A total of 100 feed samples (200 g each), were collected for mycological examination.
These samples included poultry feeds and feed stuffs served for animals, and poultry.
Samples were collected from different farms in Cairo.
Samples were stored at the refrigerator until examination.
Methods and Results
1. Isolation and Identification of moulds:
Macroscopic examination
Microscopic examination
Feed
Samples
Incidence Total Colony Count (TCC)
Colony/gm
No. of
samples
No of
positive
samples
% Maximum
TCC
Minimum
TCC
Average
TCC
Poultry
feeds 20 18 90 3.6 × 103 5.0 × 102 1.43 × 103
Yellow
corn 20 20 100 3.5 × 103 6.0 × 102 1.46 × 103
White corn 20 20 100 2.8 × 103 2.5 × 102 1.37 × 103
Wheat 20 18 90 2.6 × 103 4.5 × 102 1.21 × 103
Soya
beans 20 16 80 3.1 × 103 4.5 × 102 9.37 × 102
Total 100 92 92 - - -
0
500
1000
1500
2000
2500
3000
3500
4000
Poultry feeds yellow corn white corn wheat soya beans
Co
lon
y/g
m
Feed samples
Maximum
Minimum
Average
Samples
Genera of moulds
Aspergillus species Penicillium species Fusarium species
Maximum Minimum Average Maximum Minimum Average Maximum Minimum Average
Poultry
feeds 2.60 × 103 2.50 × 102 8.73 × 102 9.00 × 102 2.00 × 102 2.80 × 102 8.00 × 102 2.00 × 102 0.90 × 102
Yellow
corn 2.40 × 103 2.00 × 102 8.95 × 102 9.00 × 102 1.00 × 102 1.90 × 102 1.00 × 103 2.00 × 102 1.58 × 102
White
corn 1.50 × 103 1.50 × 102 8.05 × 102 8.00 × 102 2.00 × 102 1.70 × 102 8.00 × 102 1.50 × 102 1.88 × 102
Wheat 2.10 × 103 3.00 × 102 8.15 × 102 7.00 × 102 2.00 × 102 2.25 × 102 6.00 × 102 3.00 × 102 0.65 × 102
Soya
beans 2.30 × 103 2.50 × 102 6.37 × 102 8.00 × 102 4.00 × 102 1.85 × 102 2.00 × 102 2.00 × 102 0.10 × 102
Colony count of main genera of fungi isolated from feed samples (colony/gm)
0
500
1000
1500
2000
2500
3000
Poultryfeeds
Yellowcorn
Whitecorn
Wheat Soyabeans
Co
un
t (c
olo
ny
/gm
)
Feed samples
Maximum
Minimum
Average
Colony count of Aspergillus species in feed samples
0
100
200
300
400
500
600
700
800
900
Poultryfeeds
Yellowcorn
Whitecorn
Wheat Soyabeans
Co
un
t (c
olo
ny
/gm
)
Feed samples
Maximum
Minimum
Average
Colony count of Penicillium species in feed samples
0100200300400500600700800900
1000
Poultryfeeds
Yellowcorn
Whitecorn
Wheat Soyabeans
Co
un
t (c
olo
ny
/gm
)
Feed samples
Maximum
Minimum
Average
Colony count of Fusarium species in feed samples
Isolates
Poultry feeds Yellow corn White corn Wheat Soya beans Total
20 20 20 20 20
No. % No. % No. % No. % No. % No. %
Aspergillus
spp. 17 85 18 90 19 95 16 80 16 80 86 86
Penicillium
spp. 10 50 11 55 8 40 10 50 7 35 46 46
Fusarium spp. 4 20 6 30 8 40 3 15 1 5 22 22
Rhizopus 5 25 3 15 5 25 4 20 4 20 21 21
Mucor 4 20 5 25 6 30 4 20 3 15 22 22
Cladosporium 2 10 2 10 3 15 2 10 1 5 10 10
Alternaria 2 10 1 5 2 10 2 10 1 5 8 8
Prevalence of mould species in feed samples
0
10
20
30
40
50
60
70
80
90
Aspergillusspp.
Penicilliumspp.
Fusarium spp. Rhizopus Mucor Cladosporium Alternaria
Inci
den
ce (
%)
Mould species
prevalence
Prevalence of mould species in feed samples
Isolates
Poultry feeds Yellow corn White corn Wheat Soya beans Total
20 20 20 20 20
No. % No. % No. % No. % No. % No. %
Aspergillus
flavus 11 55 9 45 10 50 8 40 9 45 47 47
Aspergillus
niger 6 30 10 50 9 45 10 50 9 45 44 44
Aspergillus
fumigatus 2 10 3 15 3 15 1 5 2 10 11 11
Aspergillus
ochraceus 3 15 3 15 4 20 3 15 2 10 15 15
Aspergillus
terreus 2 10 2 10 2 10 3 15 2 10 11 11
Aspergillus
parasiticus 1 5 2 10 1 5 1 5 0 0 5 5
Aspergillus
glaucus 3 15 2 10 3 15 2 10 3 15 13 13
Aspergillus
candidus 3 15 1 5 1 5 2 10 1 5 8 8
Prevalence of Aspergillus species in feed samples
0
5
10
15
20
25
30
35
40
45
50
Aspergillusflavus
Aspergillusniger
Aspergillusfumigatus
Aspergillusochraceous
Aspergillusterreus
Aspergillusparasiticus
Aspergillusglaucus
Aspergilluscandidus
inci
de
nce
(%)
Aspergillus species
prevalence
Prevalence of Aspergillus species in feed samples
Samples incidence
No. %
Poultry
feeds 11 55
Yellow corn 9 45
White corn 10 50
Wheat 8 40
Soya beans 9 45
Total 47 47
0
10
20
30
40
50
60
Poultryfeeds
Yellowcorn
White corn Wheat Soya beans
Inci
de
nce
(%
)
Feed samples
prevalence
Prevalence of suspected aflatoxigenic Aspergillus flavus in feed samples
Samples Incidence of Aspergillus niger Incidence of Aspergillus ochraceous
No. % No. %
Poultry feeds 6 30 3 15
Yellow corn 10 50 3 15
White corn 9 45 4 20
Wheat 10 50 3 15
Soya beans 9 45 2 10
Total 44 44 15 15
0
5
10
15
20
25
30
35
40
45
50
Poultryfeeds
Yellowcorn
Whitecorn
Wheat Soyabeans
Inci
de
nce
(%
)
Feed samples
prevalence
0
5
10
15
20
Poultryfeeds
Yellowcorn
Whitecorn
Wheat Soyabeans
Inci
de
nce
(%
)
Feed samples
prevalence
Prevalence of suspected ochratoxigenic Aspergillus spp. (Aspergillus niger and Aspergillus ochraceus) in feed samples
2. Screening of isolated strains of A. flavus for aflatoxin B1 production (Bauer et al., 1983). 3. Estimation of aflatoxin B1
Qualitative estimation of aflatoxin B1 by thin layer chromatography (T.L.C.), (AOAC, 1975). Quantitative estimation of aflatoxin B1 by a fluorometeric method according to AOAC (1990)
Source of
suspected
aflatoxigen
ic A.flavus
No. of
tested
isolates
No. of
aflatoxigen
ic isolates
% of
aflatoxigen
ic isolates
Levels of aflatoxins produced (ppb)
Maximum Minimum Mean
Types of
produced
aflatoxins
Poultry
Feeds 11 9 81.8 2200 250 600 B1,B2
Yellow
corn 9 8 88.9 3000 150 750 BI
White corn 10 7 70.0 2500 140 450 BI
Wheat 8 4 50.0 1200 300 550 B1
Soya bean 9 5 55.6 4400 20 170 B1, B2, G1
, G2
Total 47 33 70.2 - - - -
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Poultry Feeds Yellow corn White corn Wheat Soya bean
Am
ou
nt
of
afl
ato
xin
s (p
pb
)
Feed samples
Maximum
Minimum
Mean
4. Screening of isolated strains of A. ochraceous and A. niger for ochratoxin production
5. Estimation of ochratoxin A
Qualitative estimation of ochratoxin A by thin layer chromatography (T.L.C.) (Vender Merwe et al., 1965 and Scott and Hand, 1967)
Quantitative estimation of ochratoxin A by a fluorometeric method from fungal culture filtrates according to AOAC (1990)
Source of
suspected
ochratoxigenic
isolates of
Aspergillus niger
No. of
tested
isolates
No. of
ochratoxigeni
c isolates
% of
ochratoxigeni
c isolates
Levels of ochratoxigenic produced (ppb)
Maximum Minimum Mean
Poultry Feeds 6 3 50.0 800 120 550
Yellow corn 10 6 60.0 250 100 150
White corn 9 5 55.6 300 150 160
Wheat 10 4 40.0 600 40 100
Soya bean 9 4 44.4 500 150 350
Total 44 22 50.0 - - -
Source of suspected
ochratoxigenic
isolates of
Aspergillus
ochraceus
No. of
tested
isolates
No. of
ochratoxigenic
isolates
% of
ochratoxigenic
isolates
Levels of ochratoxigenic produced (ppb)
Maximum Minimum Mean
Poultry Feeds 3 3 100.0 1200 400 700
Yellow corn 3 2 66.6 900 300 600
White corn 4 3 75.0 800 400 550
Wheat 3 2 66.6 600 200 400
Soya bean 2 2 100.0 400 200 300
Total 15 12 80.0 - - -
0
100
200
300
400
500
600
700
800
PoultryFeeds
Yellowcorn
Whitecorn
Wheat Soyabean
Am
ou
nt
of
och
rato
xin
(p
pb
)
Feed samples
Maximum
Minimum
Mean
0
200
400
600
800
1000
1200
PoultryFeeds
Yellowcorn
Whitecorn
Wheat Soyabean
Am
ou
nt
of
och
rato
xin
(p
pb
)
Feed samples
Maximum
Minimum
Mean
Levels of ochratoxin A produced by A. ohraceus isolated from feed samples
Levels of ochratoxin A produced by A. niger isolated from feed samples
6. Molecular Identification and of Aspergillus spp. Isolates by using polymerase chain reaction (PCR) technique: Preparation of fungal agents
Extraction of DNA according to QIAamp DNeasy Plant Mini kit instructions
Amplification of Internal Transcribed Spacer 1 gene of Aspergillus species
DNA Molecular weight marker
Agarose gel electrophoreses (Sambrook et al., 1989) with modification
All genomic DNAs used in this work were tested for suitability for PCR amplification using primers ITS1 and ITS4 (White et al., 1990).
Amplify the Internal transcribed spacer region (ITS) region in Aspergillus.
ITS amplicons from Aspergillus species ranged in size from 595 to 613 bp.
Comparison of reference strains and GeneBank sequences demonstrated that both ITS 1 and ITS 2 regions were needed for accurate identification of Aspergillus at the species level. Intraspecies variation among clinical isolates and reference strains were minimal.
The PCR test was applied on 16 fungal isolates which were morphologically and microscopically identified as A. flavus (5 isolates), A. ochraceous (6 isolates) and A. niger (5 isolates) selected from the isolated mycotoxigenic Aspergillus species according to their mycotoxin profile.
Primer Sequence
(5'-3')
Amplified
product Reference
ITS1-F 5'-TCCGTAGGTGAACCTGCG G-3'
570-600bp (Mirhendi et al., 2007)
ITS4-R 5'-TCCTCCGCTTATTGATATGC-3'
Target Primary
denaturation
Secondary
denaturation Annealing Extension No. of cycles
Final
extension
ITS-region 95°C for 5 min.
94°C for 30 sec.
56°C for 45 sec.
72°C for 1 min. 35
72°C for
7 min.
Oligonucleotide primers used for molecular identification and sequencing of Aspergillus spp.
Thermal profile used
600 bp
100 bp
600 bp
100 bp
The PCR amplification of the ITS region of Aspergillus spp. (1) 100bp ladder, (2-6) A. niger isolates.
The PCR amplification of the ITS region of Aspergillus spp. (Ladder) 100bp ladder, (1-6) A. ochraceus isolates.
The PCR amplification of the ITS region of Aspergillus spp. (1) 100bp ladder, (2-6) A. flavus isolates.
7. Sequencing of the DNA of the Amplified Internal Transcribed Spacer gene to identify the species of the isolated Asperigillus isolates Purification of the PCR Products (QIAquik PCR
product purification protocol, Using QIAquick PCR Product extraction kit. (Qiagen Inc. Valencia CA)
Sequencing reaction (Instruction of the manufacture Big dye Terminator V3.1 cycle sequencing kit)
Temperature Time Cycle
96˚ C 1 min 96˚ C
96˚ C 10 sec 25 Cycle
50˚ C 5 sec
60˚ C 2 min
Thermal profile used
Amount Reagent
2µl Big dye terminator v.3.1
1µl Primer
From 1 to 10 µl Template according to quality
of band and concentration of DNA
Complete till to total volume become 20µl Deionized water or PCR grade
Water
20µl (Mix well, spin briefly) Total volume
8. Molecular Identification of aflatoxins genes
1. Preparation of fungal agents
2. Extraction of DNA According to QIAamp DNeasy Plant Mini kit instructions
3. Real time PCR for detection of Aflatoxin by using nor-1 gene
a) Oligonucleotide primers and probe encoding for nor-1 gene used in Real time PCR for detection of Aflatoxin (Passone et al. 2010)
b) Preparation of PCR Master Mix for nor-1 gene real time PCR (According to QuantiTect probe RT-PCR kit handbook)
Amplified product (bp) Sequence
(5'-3') Primer/ probe
66 bp
GTCCAAGCAACAGGCCAAGT nortaq-1
TCGTGCATGTTGGTGATGGT nortaq-2
FAM-TGTCTTGATCGGCGCCCG-TAMRA Norprobe
Component Volume/reaction
2x QuantiTect Probe RT-PCR Master Mix 12.5 μl
Forward primer (50 pmol) 0.5 μl
Reverse primer (50 pmol) 0.5 μl
Probe (30 pmol) 0.125 μl
Rnase Dnase free water 5.25 μl
Template DNA 6 μl
c) Cycling conditions of primers and probes
Step Number of cycles Time Temperature
1. Primary denaturation
and activation of Taq
DNA polymerase.
1 cycle 4 min. 94˚C
2. Cycling 40 cycles
A. Secondary
denaturation 30 sec. 95˚C
B. Primer annealing 30 sec. 53˚C
C. Extension 20 sec. 72˚C
Results of using Oligonucleotide primers encoding for nor-1 gene for detection of Aflatoxigenic strains by using Real time PCR technique
Isolate Mycotoxin
profile
Aflatoxin
results using
nor-1 gene
CT
1 Negative Negative -
2 Negative Positive 32.33
3 Positive Positive 24.54
4 Positive Positive 26.64
5 Positive Positive 27.70
4. Molecular Identification of aflatoxins by detection of OmtB gene using conventional PCR technique
Preparation of fungal agents
Extraction of DNA According to QIAamp DNeasy Plant Mini kit instructions
Preparation of conventional PCR Master Mix
according to Emerald Amp GT PCR mastermix (Takara) Code No. RR310A kit
Component Volume/reaction
Emerald Amp GT PCR mastermix (2x premix) 12.5 μl
PCR grade water 4.5 μl
Forward primer (20 pmol) 1 μl
Reverse primer (20 pmol) 1 μl
Template DNA 6 μl
Amplification of target gene 1. Oligonucleotide primers encoding for OmtB gene used
Gene Primer Sequence
(5'-3')
Amplified
product Reference
OmtB OmtB.F ATGTGCTTGGGXTGCTGTGG
611 bp Rahimi et al.,
2008 OmtB.R GGATGTGGTYATGCGATTGAG
X= inosine which was used in forward primer which binds to all nucleotides except guanine Y= in reverse primer, Y was replaced with C+T
2. Cycling conditions of the different primers during conventional PCR technique
Target Primary
denaturation
Secondary
denaturation Annealing Extension No. of cycles
Final
extension
Aflatoxin
(omtB gene)
95˚C
10 min.
94˚C
45 sec.
55˚C
45 sec.
72˚C
45 sec. 35
72˚C
10 min.
DNA Molecular weight marker
Agarose gel electrophoreses (Sambrook et al., 1989) with modification
Isolate Mycotoxin profile PCRresults using
omtB.F/omtB.R primers
1 Negative Negative
2 Negative Negative
3 Positive Positive
4 Positive Positive
5 Positive Positive
Neg Pos 5 L 4 3 2 1
9. Molecular Identification of ochratoxingeic Aspergillus spp.
Preparation of fungal agents
Extraction of DNA According to QIAamp DNeasy Plant Mini kit instructions
Preparation of conventional PCR Master Mix
according to Emerald Amp GT PCR mastermix (Takara) Code No. RR310A kit
Amplification of target gene
Methods (Cont.)
Gene Primer Sequence
Amplif
ied
produ
ct
Target Reference
rRNA gene [ITS-
region (ITS1-
5.8S-ITS2
sequence)]
OCRA1 CTTCCTTAGGGGTGGCACAGC
400 bp A. Ochraceous
(species specific)
Patiňo et al.,
2005 OCRA2 GTTGCTTTTCAGCGTCGGCC
Polyketide
Synthase
Aopks1 CAGACCATCGACACTGCATGC
549 bp A. ochraceous
A. niger
Reddy et al.,
2013 Aopks2 CTGGCGTTCCAGTACCATGAG
1. Oligonucleotide primers sequences to detect ochratoxigenic Aspergillus species (A. ochraceous and A. niger)
Target Primary
denaturation
Secondary
denaturation Annealing Extension
No. of
cycles
Final
extension
Ochratoxin ( ITS-
region) (ITS1-
5.8S-ITS2
sequence) (OCRA)
95˚C
5 min.
94˚C
30 sec.
50˚C
30 sec.
72˚C
1 min. 35
72˚C
10 min.
Ochratoxin
(Polyketide
Synthase
gene)(Aopks)
95˚C
10 min.
94˚C
45 sec.
55˚C
45 sec.
72˚C
45 sec. 35
72˚C
10 min.
2. Cycling conditions of the different primers during conventional PCR technique
DNA Molecular weight marker
Agarose gel electrophoreses (Sambrook et al., 1989) with modification
Isolate Apergillus strain Mycotoxin profile PCR results using
OCRA1/OCRA2 primers
6 A. ochraceous Positive Positive
7 A. ochraceous Positive Positive
8 A. ochraceous Positive Positive
9 A. ochraceous Negative Negative
10 A. ochraceous Negative Positive
11 A. ochraceous Negative Negative
12 A. niger Positive Negative
13 A. niger Positive Negative
14 A. niger Positive Negative
15 A. niger Negative Negative
16 A. niger Negative Negative
16 15 14 13 12 Pos L Pos 11 10 9 8 7 6
Sample Apergillus strain Mycotoxin profile PCR Results using
Aopks1/ Aopks2 primers
6 A. ochraceous Positive Positive
7 A. ochraceous Positive Positive
8 A. ochraceous Positive Positive
9 A. ochraceous Negative Negative
10 A. ochraceous Negative Negative
11 A. ochraceous Negative Negative
12 A. niger Positive Positive
13 A. niger Positive Negative
14 A. niger Positive Positive
15 A. niger Negative Negative
16 A. niger Negative Negative
16 15 14 13 12 Pos L 11 10 9 8 7 6