Application of PCR for the Detection of Universal Fungi in ...Infants are more susceptible to the effects of mycotoxins, due to their weak immune system. As most mycotoxins are toxic

International Journal of Scientific and Research Publications, Volume 8, Issue 6, June 2018 527 ISSN 2250-3153

http://dx.doi.org/10.29322/IJSRP.8.6.2018.p7814 www.ijsrp.org

Application of PCR for the Detection of Universal Fungi in Infant Food Products Sold in Sri Lanka

Reema Sameem

Undergraduate BMS School of Science Sri Lanka

DOI: 10.29322/IJSRP.8.6.2018.p7814 http://dx.doi.org/10.29322/IJSRP.8.6.2018.p7814

Abstract- Infant food products were collected from supermarkets in Sri Lanka and investigated for contamination by fungi. The objective of this study was to develop a novel DNA extraction procedure and a PCR assay for the detection of fungal presence which leads to contamination of infant foods. Mycotoxins are secondary metabolites of many phytopathogenic and food spoilage fungi. The presence of mycotoxins in the food chain is of high concern for human health due to the ability of these compounds to induce severe toxic effects. Thus the rapid identification of fungi would be desirable, such that early intervention could limit the amounts of contaminated food products in the market. The specificity and sensitivity of the PCR procedure, combined with its speed, reliability, cost-effectiveness and user-friendly protocols for its application offer several advantages for the identification of fungi in food commodities. Thus far, no single reported DNA extraction technique is suitable for the efficient extraction of DNA from the entire fungal species present in food matrices. Two CTAB DNA extraction methods were compared and optimal extraction was achieved. The ITS1, ITS2 regions and the 5.8S rDNA region of the fungi were amplified by using universal primers ITS1 and ITS4. Samples of infant food displayed bands between 580-700 bp lengths which is an alarming indicator for the possibility of the presence of mycotoxins in infant food samples. In conclusion, this protocol is reproducible and generates good yield and quality DNA for molecular analysis of infant food using PCR. Index Terms- fungi, infant food products, Sri Lanka, mycotoxins, PCR, primer

I. INTRODUCTION 1.1 Global occurrence of mycotoxins ungi are ubiquitous microorganisms that are associated with the spoilage and deterioration of food commodities. Consumption of

unsafe, contaminated food leads to food-borne diseases which are a significant cause of morbidity and mortality worldwide. Each year as many as 600 million, almost 1 in 10 people around the world, are reported ill after consumption of contaminated foods. Out of which, 420 000 people die, including 125 000 children under the age of 5 years [1].

Figure 01. Prevalence of mycotoxins worldwide [2].

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http://dx.doi.org/10.29322/IJSRP.8.6.2018.p7814

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Mycotoxins are secondary metabolites produced by filamentous fungi which, when ingested by humans, causes a toxic response, known as mycotoxicosis. The primary genera responsible for mycotoxins in the human food chain are Aspergillus, Fusarium and Penicillium [3]. The toxicity and carcinogenicity of these mycotoxins is a serious concern globally (Figure 01). Consequently, contaminated foods (Table 01) constitute a health hazard to human consumption [4].

Table 01. Important toxigenic species and mycotoxins in food commodity [5].

Species Commodity Mycotoxin Penicillium expansum Fruit Juice Patulin Aspergillusflavus,A. Dairy Products Aflatoxin M1 parasiticus P. verrucosum, A. ochraceus Porcine based products, Ochratoxin A Beer, Coffee Fusarium graminearum, F. All Cereals Type B Trichonthecenes culmorum and Zearalenone F. poae Small grain cereal Type A Trichonthecenes F. proliferatum Maize Moniliformin F. moniliforme Maize Fumonisins

The production of mycotoxins is influenced by moisture, temperature and time. Contamination can occur throughout the food chain from the field, during harvesting, processing, storage, transportation, and consumption. Tremendous efforts are made to exclude food pathogens by using principles of Good Manufacturing Practice and Good Hygiene Practice [6]. 1.2 Chemical composition of mycotoxins There are over 300 mycotoxins of which about 20 of these are relevant to public health. The most significant mycotoxins include, aflatoxins, trichotecenes, ochratoxins, fumonisin, zearalenone, and patulin. The diverse range of chemical structures (Figure 02) of mycotoxins results in a broad spectrum of toxic effects, including hepatotoxic, neurotoxic, teratogenic, nephrotoxic and immunosuppressive effects [7]. These mycotoxins are chemically stable and therefore, although exposed to high temperature, are not degraded during processing [8].


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Figure 02. Chemical structure of common mycotoxins produced by mycotoxigenic fungi [9].

1.3 Mycotoxins and its association with infant food Baby foods are the primary source of nutrition for infants before their ability to digest other types of food [10]. The manufacture of infant food is based on dried cereals, including wheat, rice, maize, oats, millet and additional ingredients such as, dried fruits. Therefore, baby foods are rich in carbohydrates, proteins, and fats. Infants are more susceptible to the effects of mycotoxins, due to their weak immune system. As most mycotoxins are toxic in very low concentrations, infant foods must be examined at regular intervals in order to assess the hygienic quality. Various research have been carried out globally, for the identification of fungi in infant food, including in Uganda [11], Libya [12], North Africa [13], Morocco [14], Poland and East Slovakia [15].


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In Sri Lanka, studies have been carried out for the identification of fungi from medicinal plant material [16], commercially available peanuts [17], cow’s milk [18] and pepper samples [6]. Nevertheless, there is no evidence of research carried out for the detection of fungi in infant food samples. 1.4 Regulatory limits of mycotoxins To date, many international agencies have implemented universal standardization of regulatory limits for mycotoxins (Table 02). Special emphasis has been drawn to mycotoxin contamination of baby foods as infants are more susceptible to the effects of these toxins [19].

Table 02. Regulatory limits of aflatoxin subtypes and total aflatoxins in Asian countries [20].

Country Types of Maximum Food and feed commodities Aflatoxins limit (ppb) China B1 20 Maize, Peanut B1, M1 0 Infant foods Hong Kong Total 20 Peanut and Peanut Products Total 15 Other foods India B1 30 All Indonesia Total 50 Corn feed Total 35 All foods B1 20 All foods B1/total 15/20 Peanut, corn and their products M1 5 Dried milk and related products Japan Total 10 All foods B1 10 Rice Malaysia Total 35 All Philippines Total 20 Human foods B1 20 Coconut, peanut products M1 0.5 Milk Singapore Total 5 All South Korea B1 10 Grains, Cereal Products, Dried B1 0.1 fruits, Rice B2, G1 and 15 Infant Foods G2 Grains, Cereal products, Dried fruits, steamed rice and baby foods Sri Lanka Total 30 All M1 1 Infant foods B1, B2, G1 30 Groundnuts, cereals, floor and oil and G2 seeds

1.5 Identification of fungi in food commodities Due to the importance of fungi in food quality, rapid and accurate methods to identify and enumerate these contaminants in food matrices are essential. Traditionally, food borne fungi were identified by culture techniques which required the use of selective microbiological media. Although effective, these techniques are exceptionally labour intensive and time consuming [21]. Over the past decade, various molecular methods based on immunological and genotypic techniques have been developed for revealing the occurrence of fungi in food matrices. Among these techniques, PCR is one of the most promising analytical tools in food microbiology due of its high specificity and sensitivity [22]. Since its development in the mid-1980s, PCR has revolutionised molecular biology. PCR allows the amplification of specific fragments of DNA from complex DNA samples (Figure 03).


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Figure 03. The Polymerase Chain Reaction [23]. The resulting PCR product can be observed after gel electrophoresis and stained with a DNA binding fluorescent dye such as ethidium bromide [24]. 1.6 The molecular target in fungi The advent of the PCR has expedited the molecular analysis of fungal genome studies. Thus far, the rDNA is the most popular DNA region targeted for the identification of fungi as rDNAs are prevalent at high copy numbers [25]. The fungal nuclear rDNA unit consists of three genes, the large subunit rRNA gene (25S), the small subunit rRNA gene (18S) and the 5.8S rRNA gene, which is separated by internal transcribed spacer (ITS) regions (Figure 04). The ITS regions, tandemly repeated many times in the genome, are highly variable and often used to distinguish taxonomic groups. The ITS region is the formal fungal barcode and the most sequenced fungal marker [26].

Figure 04. The structure of a repeat unit of the nuclear rDNA cluster in fungi [27]. Universal primers are available which allow the ITS regions to be amplified using conserved sequences from within the rDNA gene sequences. Coding regions of the 18S, 5.8S, and 25S nuclear rRNA genes evolve slowly, and are relatively conserved among fungi. Between the coding regions are the ITS1 and ITS2 regions which evolve more rapidly. Therefore, the ITS is more useful for the development of specific oligonucleotide primers [28]. In this study, the ITS1, ITS2 regions and the 5.8S rDNA region of the fungi were amplified by using universal primers ITS1 and ITS4 (Figure 05).


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Figure 05. Map of typical fungi rRNA genes illustrating the positions of variable regions and the universal primers to amplify these regions. Indicated in grey conserved regions and white variable regions of DNA. The aim of this study was to extract fungal DNA and to optimize the PCR reaction in order to detect the presence of fungi in infant food products sold in Sri Lanka, by using molecular techniques. Moreover, this study was designed to develop a novel DNA extraction procedure and a PCR assay for the detection of fungi involved in contamination of infant food samples which have been claimed to be sterile by the manufacturer.

II. MATERIALS 2.1 Consumables: - Micro-centrifuge tubes - Micro-centrifuge tube racks - Micropipettes (10 µL, 20 µL, 200 µL and 1000 µL) - Gloves - Pipette tips (Blue and Yellow) - Permanent marker -Magnetic Bar - Gel casting tray - Parafilm - Gel combs - -Measuring Cylinder - Tube racks 2.2 Reagents: - 70 % Ethanol - CTAB - CTAB precipitation solution - NaCl - Chloroform - - Agarose Powder - Forward Primer (ITS 1) -Reverse Primer (ITS 4) - Taq DNA polymerase - DNA ladder -Isopropanol -Food Sample -dNTPs –PCR Buffer -Nuclease Free water -TBE buffer - Ethidium Bromide -Sterile deionized water


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2.3 Instrumentation

Table 03. Overview of instrumentation

Instrument Model Manufacturer

Centrifuge NF 048 NÜVE, Turkey

Dry Bath Incubator #8580.35 Equitron, India

Electric Balance D-72336 KERN and Sohn, Germany

Electric Table Lamp Philips

Electrophoresis power supply YERCAUD 636 601 BIOTECH R & D Laboratories, India

Electrophoresis power supply MC- 01 TARSONS, The Science house, India

Gel documentation system (E- 4466611 Life technologies, Invitrogen, USA Gel Imager)

Magnetic Stirrer SPINOT, SINGHLA Scientific Industries, India

Micro-centrifuge machine DW-41 QUALITRON, INC., Korea

Midi Submarine 7050 TARSONS, The Science house, India Electrophoresis Unit

PCR work station hood Bangalore Genei Pvt. Ltd., India

Post- PCR Refrigerator USHA, India

Pre- PCR Refrigerator LG

Thermal Cycler 2720 Applied Biosystems by Life technologies, USA


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III. METHODOLOGY 3.1 Sample collection: Six samples of varying brands of infant food (Table 04) were collected from local stores and retailers. The samples which consist of imported and locally produced products were stored at room temperature in air tight jars. The samples purchased were within the expiration date and were examined molecularly to determine the presence of fungi.

Table 04. Outline of the infant food samples

No. Product Main Ingredients Country of Origin

1 Brand A Apple, Oatmeal and Cinnamon Extract Australia

2 Brand B Papaya and Mandarin Sri Lanka

3 Brand C Banana, Lime Juice and Vitamin C Sri Lanka

4 Brand D Pumpkin, Carrot, Red rice, Dhal, Lime juice Sri Lanka and Centella Asiatica(Gotukola)

5 Brand E Apple, Blueberry, Rice, Antioxidant ascorbic Germany acid and carrot juice extract

6 Brand F Carrot, Red rice and Lime Juice Sri Lanka 3.2 DNA Extraction: Two CTAB DNA Extraction procedures were compared, one omitting proteinase K and RNase A treatment, and one following the standard protocol. Method 01 A portion of 100 mg of homogenized sample was transferred to a 1.5 ml micro-centrifuge tube. Thereafter, 1000 µL CTAB extraction buffer (100 mM Tris-Cl, 2g/100 mL CTAB, 1.4 M NaCl, 20 mM EDTA – See Appendix A) was added to the micro-centrifuge tube, and mixed well. The mixture was incubated at 65 °C for 60 minutes. Following which the suspension was centrifuged for 5 minutes at 14000 rpm. On to a new micro-centrifuge tube, 500 µL of supernatant was transferred and extracted with 200 µL of chloroform, mixed well, and centrifuged for 5 minutes at 14000 rpm. The supernatant was then transferred to a new micro-centrifuge tube, and mixed with double volume of CTAB precipitation solution (5 g.L–1 CTAB, 0.04 M NaCl – See Appendix A). The micro-centrifuge tube was incubated for 60 minutes at room temperature. Upon centrifugation for 5 minutes at 14000 rpm, the supernatant was discarded and the precipitate was dissolved in 350 µL 2 M NaCl. Thereafter, 350 µL of chloroform was added, mixed for30 seconds and then centrifuged for 5 minutes at 14000 rpm. The supernatant was mixed with equal volume of isopropanol and stored at –20 °C overnight. Thereafter, the micro-centrifuge tube was centrifuged for 5 minutes at 14000 rpm. The precipitate DNA was hydrated in 100 µL of DNA grade water and stored at –20 °C. Method 02 On to a 1.5 mL micro-centrifuge tube, 100 mg of homogenized sample was transferred. To this, 300µL of sterile deionized water was added and mixed well. Thereafter, 500 µL of CTAB buffer was added and mixed well. Following the addition of 10µL of Proteinase K (20 mg/ml), the micro- centrifuge tube was mixed and incubated at 65 °C for 60 minutes. Thereafter, 2 µL of Rnase A (10mg/ml) was added, mixed well and incubated at 65 °C for 5 minutes. The suspension was then centrifuged at 13000 rpm for 5 minutes.


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The supernatant was transferred, on to a new micro-centrifuge tube containing 500 µL chloroform and mixed well for 30 seconds. Thereafter, the micro-centrifuge tube was centrifuged for 5 minutes at 13 000 rpm. This step was repeated once more. The upper layer was then transferred to a new micro-centrifuge tube. To this, 2 volumes of CTAB, precipitation solution was added and mixed well by pipetting. The micro-centrifuge tube was then incubated at room temperature for 60 minutes. Following which, the suspension was centrifuged for 5 minutes at 13000 rpm. The supernatant was discarded and the precipitate was dissolved in 350 µL NaCl (2 M). This was followed with the addition of 350 µL of chloroform and mixed well for 30 seconds. The micro-centrifuge tube was centrifuged at 13000 rpm for 5 minutes. Thereafter, the supernatant was transferred to a new micro-centrifuge tube and 0.6 volumes of Isopropanol was added, and mixed well. The suspension was centrifuged at 13000 rpm for 10 minutes. The supernatant was discarded. On to the micro-centrifuge tube, 500µL of 70% ethanol was added and mixed carefully. Thereafter, the tube was centrifuged at 13000 rpm for 5 minutes and the supernatant was discarded. The pellets were dried under a lamp and re-dissolved in 100 µL of nuclease free water. In order to compare the two extraction methods, DNA from three samples – Fungi plate (Positive Control), Nuclease free water (Negative Control) and Infant food sample were extracted using both Method 01 and Method 02. Subsequent extractions were carried out using the most efficient extraction procedure – Method 02. 3.3. PCR Assay The PCR reaction mixture (Appendix B) of 50 μl contained, 5 μl of 10x PCR buffer, 2 μl (10 mM) of dNTPs, 3 μl each of 10 μM of ITS 1 and ITS 4 primers, 0.5 μl of Taq DNA polymerase, 31.5 μl of sterile distilled water and 5μl of template DNA. The PCR mix was prepared for a total volume of 450 μl. One each for the six infant food samples, negative control, positive control and extra volumes to compensate for pipetting errors. The PCR master mixture was aliquoted into 10 PCR tubes containing 45 μl each. The DNA samples were obtained from -20 °C, incubated at 65 °C for 5 minutes and then centrifuged at 12 000 g for 30 seconds. On to each PCR tube, 5 μl of respective DNA samples were added. Nuclease free water was added to the negative control in place of DNA. The ITS1, ITS2 regions and the 5.8S rDNA region of the fungi were amplified by using universal primers ITS1 and ITS4 (Table 05) for forward and reverse amplification, respectively.

Table 05. Primers used for PCR amplification of fungal DNA

Table 06. Cyclic Parameters for PCR

Primer name Primer direction Primer sequence (5’ 3’) Tm ºC GC%

ITS1 Forward TCCGTAGGTGAACCTGCGG 61.7 63.16

ITS4 Reverse TCC TCC GCT TAT TGA TAT GC 56.4 45.00

Steps Temperature Time Initial denaturation 94ºC 5 minutes

Denaturation 94ºC 30 seconds

Annealing Cycles 40 52ºC 1 minute

Extension 72 ºC 1 minute

Final hold 72ºC 5 minutes


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3.4: Gel Electrophoresis The amplified products were subjected to electrophoresis on a 2% agarose gel (0.6 g of agarose in 30 ml 5x TBE buffer –Appendix C), and stained with 10 μl of ethidium bromide in the medium of 5x TBE buffer, and 10 μl of PCR product were loaded into the wells. A 100-bp DNA ladder (GeneShun Biotech) was run concurrently with amplicons for sizing of the bands. The gel was run at a voltage of 80 V for 60 minutes. Thereafter, the gel was visualized under UV trans-illuminator.

IV. RESULTS 267 268 Figure 6. Agarose gel electrophoresis of PCR Products in order to compare two CTAB extraction methods. Lane M - 100 bp DNA ladder (GeneShun Biotech), Lane 1 - Fungi plate, Method 01, Lane 2 - Infant food sample, Method 01, Lane 3 - Nuclease free water, Method 01, Lane 4 - Fungi plate, Method 02, Lane 5 - Infant food sample, Method 02, Lane 6 - Nuclease free water, Method 02, Lane 7 - Positive Control.


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293 294

Figure 7. Agarose gel electrophoresis of PCR products amplified from DNAs extracted from infant food samples using universal fungal primers ITS1 and ITS4. Lane M -100 bp DNA ladder (GeneShun Biotech), Lane 1 - Positive Control (Fungi DNA), Lane 2 - Brand A, Lane 3 - Brand B, Lane 4 - Brand C, Lane 5 - Brand D, Lane 6- Brand E, Lane 7- Brand F, Lane 8- Negative Control (Nucleas Free water).

V. DISCUSSION There is an increasing need for more rapid and sensitive methods to detect food borne fungi. Due to their high specificity, sensitivity and rapidity, PCR has been successfully applied for food analysis. In the present study, PCR assays were used to semi-quantitatively detect the amount of fungal DNA in infant food by staining with ethidium bromide and evaluation on agarose gel. The structure of the fungal cell wall is highly complex and consist of thick layers of chitin, (1–3)- β-d-glucan, (1,6) β-glucans, peptides and lipids. This physical characteristic of fungi has prevented the development of a universal fungal DNA extraction method. Therefore, there is no single method of cell lysis which is appropriate for all fungal species [30]. The efficiency of the DNA extraction step is critical for the successful amplification. The sensitivity of the PCR depends of the efficiency of the method used to extract fungal DNA. Furthermore, infant food products have been subjected to processing, including mechanical, chemical, enzymatic or thermal treatment, affecting the integrity of DNA. Thus, DNA isolation methods for infant food samples must provide sufficient removal of food additives, and preservatives, as they might interfere with downstream applications [31]. To obtain optimal detection of fungi by PCR, it is necessary to eliminate substances inhibitory to amplification such as polysaccharides, lipids or phenolic compounds that affect PCR amplification, especially with complex food matrices such as infant foods. As these can lead to ambiguous results [32].


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In this study, CTAB extraction method was used as it is cost-effective and provides an optimal yield of DNA. The CTAB extraction buffer contains chaotropic salts and mixtures of detergents. CTAB is a cation-active surfactant assisting in cell wall disruption and removal of polysaccharides, thus facilitating the release of nucleic acid [33]. Research demonstrates that the method of cell wall disruption considerably influences the effectiveness of DNA isolation. For downstream PCR applications, the extraction method should maximize yield and not affect the quality and purity of DNA as contaminants carried over from preceding extraction steps can significantly reduce the sensitivity of the PCR. When comparing the two CTAB extraction methods (Figure 6), method 01 (Lanes 1, 2 and 3) produced bands of greater intensity than method 02 (Lanes 4, 5 and 6). However, a band was not observed corresponding to the fungi plate in method 01 (Figure 6, Lane 1). The absence of DNA in the PCR reaction mixture could be due to the insufficient cell wall disruption. Another possible cause is DNA loss during the extraction or purification steps. Moreover, it could be due to inhibitors co- extracted with the sample DNA, which prevented the amplification of the target DNA. The presence of bands corresponding to nuclease free water (Figure 6, Lanes 3 and 6), suggests that contamination had occurred. This could be due to exposure of DNA grade water to air which could have introduced fungal DNA or due to extraction reagents being contaminated. Hence the extraction on nuclease free water was repeated and a band was not observed (Figure 7, Lane 8). Method 02 was used for subsequent extractions, due to its efficiency, and the ability to complete the extraction within a reasonable time frame of 5 hours, compared to method 01 which had overnight incubation steps and therefore, prolonged the extraction process. Furthermore, the time constraints of the project contributed to this choice. The digestion of proteins was achieved by proteinase K, while RNase A enzyme was used to remove RNA. For washing and purifying DNA, isopropanol and ethanol were used. Additionally, 70% Ethanol removes residual CTAB, salt and other contaminants. Chloroform was used to extract fungi DNA, as it separates proteins and polysaccharides from the DNA. Chloroform is denser than water, therefore upon centrifugation the solution separates into two distinct phases. Autoclaved distilled water was added during extraction in order to damp the sample and offer higher extraction efficiencies, by increasing penetration of the solvent into the hydrophilic material. Nuclease free water is added in order to dissolve the DNA pellet. The addition of NaCl during extraction neutralizes the negative charges of DNA. Negatively charged phosphates on the DNA causes molecules to repel each other. The Na+ ions form an ionic bond with the negatively charged phosphates on the DNA, allowing the DNA molecules to come together. EDTA is a chelating agent that binds magnesium ions, which is a necessary cofactor for most nucleases. This is important as the DNA must be protected from the endogenous nucleases. The agarose gel electrophoresis PCR products, positive control displayed a band correspondin to 580 bp (Figure 6, Lane 7 and Figure 7, Lane 1). Infant food brands A, B, C, D, and F (Figure 7 – Lanes 2-5 and 7) displayed bands of 700 bp. While brand E displayed a band at 580 bp (Figure 7- Lane 6). Infant food brand C (Figure 7 – Lane 04) displayed two band of size 650 bp and 700 bp. The presence of two distinct bands in brand C indicates that more than one type of fungi could be present. These products are within the expected amplified length for IST1 and ITS 4 primers. Since the PCR assay has been developed using universal fungal primers the presence of any fungi within the samples will be detected. In order to identify the specific species of fungi, DNA sequencing has to be carried out. Nevertheless, due to the time constrains of the project, DNA sequencing was not performed. Additionally, positive and negative controls of extracted DNA were introduced in each analysis in order to determine the efficiency of PCR and to detect any possible contamination. Prior to the preparation of the master mix, the PCR work station was sterilized by exposure to UV light in order to avoid contamination. Furthermore, careful separation of equipment and areas used for DNAextraction, PCR preparation, and PCR product handling; it was ensured that complications associated with contaminating DNA or PCR products were avoided. The presence of 580-700 bp length band in the agarose gel is an alarming indicator for the possibility of the presence of mycotoxins in infant food samples. Nevertheless, in the food analysis process, it is insufficient to know the presence or absence of fungi. Rather, it is the toxic properties of the fungi that indicate the route of contamination. One of the significant problems associated with the detection of fungi in infant food is the inability of PCR to distinguish the DNA of living cells from that of dead cells. Suggested further improvements could be quantification of the fungal load in the infant food samples by using real time PCR. This technique has proven to be more suitable than conventional PCR, due to its quantitative performance and greater sensitivity. The ability of real-time PCR to monitor the progress of DNA amplification in real time is accomplished by using specific chemicals and instrumentation.

VI. CONCLUSIONS In conclusion, this protocol is reproducible and generates good yields and quality DNA for molecular analysis using PCR. The quality of DNA extracted using method 02 was confirmed by the successful amplification of the ITS1, ITS2 regions and the 5.8S rDNA using ITS1 and ITS4 primers. Manufacturers of infant foods should give an extreme importance to mycotoxin content. In order to protect public health, it is essential to keep contaminants at levels toxicologically acceptable. Further studies are required to build specific PCR-based methods, which could offer continuous surveillance, as the quality of the end product depends on the precise controlling at every step of the production.


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ACKNOWLEDGMENTS I would like to express my deepest appreciation to those who provided me the prospect to complete this research project. I am grateful to Mrs. Vajirapani De Silva, Senior Scientist at Genetech School of gene technology, and Ms.Vaishnavi Krishnapillai, my supervisor, who facilitated the great opportunity of carrying out this research and for their her vital guidance and encouragement to make this project productive and efficient. Conflicts of Interest: The author declares no conflict of interest. Appendix A Preparation of Reagents CTAB Extraction Buffer CTAB (2%) 2g/ 100 ml For 50ml – 1g of CTAB NaCl (1.4 M) – Mole = C x V = 1.4 x 0.05 dm-3 = 0.07 mole Mass = Mole x Mr = 0.07 x 58.44 = 4.0908 g = 4.09 g EDTA (20mM) C1 x V1 = C2 x V2 0.5 x V1 = ( 20 x 10-3 ) x 50 V1 = 2 ml Tris-Cl (100 mM) 1 M= 121. 14 g/mol 100 x 10-3 = x x = 1.2114 g x = 1.2 g Using a electric balance 1.2g of Tric-Cl was measured. This was dissolved in 10 ml of water. C1 x V1 = C2 x V2 1 M x V1 = (100 x 10-3) x 50 V1 =5 ml From the 10 ml, 5 ml was obtained in order to prepare CTAB Extraction buffer. Using an electric balance, 1g of CTAB and 4.09 g of NaCl was measured and transferred into a conical flask. To this, 2 ml of EDTA and 5 ml of Tris Cl was added, and mixed well. The solution was transferred to a conical flask and topped up to 50 ml with de ionized water. The solution was autoclaved and stored in room temperature. CTAB Precipitation Solution For 1000 ml - 5g/l CTAB, 0.04 M NaCl Dissolve 5 g/l CTAB in 100 ml of deionized water. Top it up to 1000 ml and autoclave. Store solution at room temperature for a maximum of 6 months. For 50 ml


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CTAB 1000 ml 5 g/l 50 ml x x = 0.25 g Deionized water

1000 ml 100 ml 50 ml x x = 5 ml

NaCl – Moles = C x V = 0.04 x 0.05 dm-3 = 2 x 10-3 mol Mass = Mole x Mr = 2 x 10-3 x 58.44 = 0.1168 = 0.12 g Using an electrical balance, 0.25 g of CTAB was weighed and transferred into a conical flask. The CTAB powder was then dissolved in 5ml of autoclaved distilled water. Thereafter, a quantity of 0.12 g of NaCl was measured and added into the conical flask and mixed well. The solution was transferred to a measuring cylinder and topped up to 50 ml with autoclaved distilled water. The solution was autoclaved and stored in room temperature. Appendix B Table B1. ITS PCR (Universal Fungi Identification)

Reagent n = 1(μl) n = 10 (μl) 10 x PCR buffer 5 50 dNTPs 2 20 ITS 1 (10mM) 3 30 ITS 4 (10 mM) 3 30 Taq DNA Polymerase 0.5 5 DNA 5 5 Sterile distilled water 31.5 315 Total 50 450

Stock Primers ITS 1 and ITS 4 (50 μm) C1 x V1 = C2 x V2 50 μm x V1 = 10 x 100 V1 = 1000/50 V1 = 20 μl of stock primer + 80 μl of nuclease free water On to a 1.5 mL micro-centrifuge tube, 315 μl of Sterile distilled water was added using a P-200 micro-pipette in two steps. Thereafter, 50 μl of 10x PCR buffer was added using a P-200 micro-pipette. This was followed with the addition of 30 μl each of forward ITS 1 and reverse ITS 4 primers respectively. Finally, 5 μl of Taq DNA polymerase was added using a P- 20 micropipette.


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Appendix C Preparation of Agarose Gel The ends of the gel casting tray were sealed using masking tape. On an electric balance 0.6 g of agarose powder was weighed and transferred to a conical flask along with a magnetic bar. Using a measuring cylinder 30 ml of 5x TBE buffer was measured and then transferred to the conical flask. The conical flask was placed on a magnetic stirrer and provided with heat, until the solution was warm and colourless. The conical flask was carefully removed from the magnetic stirrer and left to cool for 10 minutes. The warm agarose solution was poured into the gel casting try and the gel comb was placed in its position. The gel was left to solidify at room temperature for 20 minutes.

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AUTHORS First Author – Reema Sameem, Undergraduate BMS School of Science Sri Lanka; [email protected]


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Application of PCR for the Detection of Universal Fungi in ...Infants are more susceptible to the effects of mycotoxins, due to their weak immune system. As most mycotoxins are toxic