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DETECTION OF ASPERGILLUS PARASITICUS ON ARTIFICIALLY INFECTED PEANUT AND CORN KERNELS BY USING PCR METHOD
Huynh Le Thao Trinh, Than Thi My Linh, Nguyen Thi Hue*
Schod of Biotechnology, Intemational Unh/ersity, Vietnam National Unhrersity in HCMC
SUMMARY
Contamination of foods and feeds by Aspergillus species and their toxic metabolites has been realized as a serious problem because of Its efiects on human and animal h ^ t h . Among the most notable mycotoxins, aflatoxins are natural carcinogenic secondary metabolites predominantly produced by A parasiticus. Currently, the detection of this fungus is tune-consuming due to its nuiphological similarities with other fringal related species. Therefore, many researchers have studied to develop more effective methods for fungal detection in foods that could help to avoid the consunq>tion of aflatoxin contaminated foods. In this study, a PCR- based method for the detection of .^. parasiticus on artificially infected dried peanut and com kernels was developed. The ii^ction of A. parasiticus on the kcraels was tested to create a condition l)emg nearly similar to naturally infected foodis. After inoculation, fungal DNA for PCR was obtained by two different methods, direcdy from the infected samples and indirectly from the fimgal ennched ones. PCR assay was successfrilly designed for the detection oiA. parasiticus in Ixith f i m ^ enriched samples and directly infected sanqiles. According to the result, PCR reaction requires at least 12 ng of extracted DNA bom 2 g of infected kernels. The obtained data showed that the directly infected sanqiles were more suitable for die fungal detection than the other ones since the advantages including time-saving, satisfied DNA quality and quantity for PCR reaction Hence this method is suggested as a preparing piDtocol for the detection of A. parasiticus on dry food in further studies.
Key words: Aspergillus parasiticus, aflatoxins, PCR method, fimgal enrichment, peanut, com kemels.
INTRODUCTION
Besides rice, com and peanut are major crops gn3wn extensively in Vietnam, with 4.6 million metric tons of com and 0.46 million mebic tons of peanut produced annually (Tran-Dinh et ai, 2009). Due to the tropical climate, grains are easily infected by molds and mycotoxins during pre and postharvest that has been recognized as a serious problem.
Mycoto)dns are secondary metabolites produced by a diverse number of microfungi. Their acute and chronic impacts on human and animal health are scientifically proven (CAST, 2003). Moreover, acconJing to ttie FAO, more than 25% of the wcHld's agricultural production is contaminated with mycotoxins. This causes economic loss. Thus, myeotoxin contamination is a trouble not only for Vietnam. Hence, most countries have adopted regulations to limit exposure to mycotoxins. Ho^'ever. the presence of mycotoxins is unavoidable, and so, testing of raw materials is required to keep our food and feed safe (Romeriab, 2012). The most important identified mycotoxins are aflatoxins. deoxynivalenol, ochratoMn A, fumonislns, zearalenone. patulin and T-2 toxin. Among them, aflatoxins have been considered as the most notable mycotoxins causing both acute and chronic food poisoning wiUi respect to potency of toxicity, carcinogenicity, and mutagenicity in human and animal. The pnmary interesting aflatoxins are aflatoxin B1, B2, GI and G2 and some of their metabolites such as aflatoxins M l . M2 and so forth. In Vietnamese foods, the range of aflatoxin concenfration was 2-640 pg/kg in 1986 and 5-41 pglkg in 1987. The frequency of occurrence of the aflatoxin positive samples was 50% In 1986 and 78% in 1987(Blaha ef ai, 1990).
Aflatoxin is secreted by many fungal spices, especially Aspergillus parasiticus and Aspergillus flavus (Hom, 2007). A.
parasiticus is a tropical and subtropical fungal species. In soils, populations of A. parasiticus are generally associated with peanut cultivation, but in low/ densities in com fields (Hom. 2003). The important differences in mycotoxin production between A. parasiticus and A. flavus are that A. parasiticus produces G as well as B aflatoxins and A. parasiticus isolates often produce aflatoxins in much higher concentrations (Hom, 2003). The conditions under which Aparasitlcus can produces aflatoxins are over the temperature range 12-40'C, water activity range 0.86 - 0.99 a*, and the pH range 3-8 or higher which are quite similar to those of A. tiavus. It appears that dry. hot conditions favor the presence of toxigenic strains of A. parasiticus (ICMSF, 1996).
Because of the toxic potential of aflatoxin, there is an uigent need to detect the fungus by highly specific, easily replaceable, and reiabveiy rapid methods. Originally, identification and detection of aflatoxigenic Aspergillus species in foods rely on microscopic or culture techniques which are laborious, time consuming and requires facilities and mycoioglcal expertise (Shapira et ai, 1996). Besides that. GC and/or HPLC with mass specfrometry (MS) have successfully been used for quantification of mycotoxins in a range of mabices such as foods. In addition, enzyme-linked immunosortjent assay (ELISA) is a rapid method for mycotoxins qualification. However, these methods are quite complicated and expensive. Recently, the application of DNA based technique PCR facilitates in vitro amplification of a target sequence and offers several advantages over fraditional methods, that permits rapid, sensitive and specific 'detection on infected food crops (Shapira e( al.. 1996). Whereas, there are still rare official procedures applied in reality 'fcr ftjngi detection in Vietnam. In previous study, we have already developed a PCR assay to detect A. parasiticus.
^however, this previous wortt was the detection of the pure fijngal DNA only.
liherefore this stijdy aims to produce a protocol to detect A. parasiticus by PCR assay using isolated DNA from infected
«imples. A suitable mettiod to isolate DNA from infected samples will be studied and be selected for the detection of A.
jSflrasrt/cus.
MATERIALS AND METHODS
Preparation of infected samples
To contiol the-specjfrcity and sensitivity of PCR assay, ttie known infected samples need to be used. For this purpose, tiie infected samples witti A parasiticus are prciduced by inoculate samples witti Aspergillus parasiticus VTCC-F-1159*
make sure ttiat samples used in detection by PCR assay are infected witti A. parasiticus. Two groups of samples wen created, com group and peanut group.
50 g peanut and 50 g com kemels were evenly sprayed witti 3.75 g of water to increase ttie moisture content to approximately 3.75%, dry basis. After moistening, all samples were exposed to UV light for 30 min. 10 peanut kemels and bwenty com kemels moistwied before were gently rolled on ttie surface of a S-day-oW culhire of A. parasiticus strair VTCC-F-1159 on malt extract agar, and then adding to each 50 g quantity of peanut and com kemels and ttiorougNj mbced. Each tteatment was replicated 5 times. Tbe samples used for fljrttier DNA exfraction were collected on ttie date before ttie fongi fully grow on surface of infected kemels For ttie negative contixil, similar conditions were applied withoiii infection of A parasiticus.
Samples treatment and DNA isolation
Tbe infected peanut and com kemels ttien undenwent hwo different processes before DNA exttaction. In direct method, ttiree. five, and ten kemels were collected randomly and washed witti 1.5 ml sodium chloride 0.8 M by shaking at 1500 rpm for 5 min at nxrni temperatore. This Is for fungal isolation followed by centritogation at 1300 rpm for 10 min. The pellet was obtained for DNA exttaction. In indirect mettiod, three or five kemels were also collected randomly and incubated on malt exhget agar for 24 hours before washed witti 1.5 ml NaCI 0.8 M. TTie enriched fungus was also harvested by centrifugation.
DNA exttaction was performed by SDS lysis buffer including grinding with sterilized sand and thermal shock This process was adopted witti some modifications ftom ttie mettiod of Plaza (Plaza et ai, 2004) and Hue (Hue et ai. 2013).
Collected pellet and sterilized glass powder mhdure were pre-ehilled at -20°C and ground to a fine powder before lh|
extinction process. Finally, for DNA evaluation. NanoDrop system was used to measure ttie concentration and ttie purit|
of DNA. The concenttatton Is expressed in unit of ng/ul and the purity is evaluated based on the ratio of absorbance all 260 nm and 280 nm.
PCR assay
The PCR assay uses a pair of primer of fonward -GGATTCGTGAGTGTCTTTAGG6, and reverse - GGTAAATGCTCCGCACAGTC that was designed based on two adjacent genes norB and eypA involved in biosynthesis of aflatoxin G in A. parasiticus, which was described in previous study. These primers used to amplify a sequence 343 bp on norS-cypA genes. 25 pi PCR reaction was performed conteinlng DNA template (4 pi for direct method and 2fjl for indirect method). 12.5 pi toptaq DNA polymerase, 1.25 pi of 10 mM each primer; and distilled water. PCR themial program indudlng 5 min initiation at 95°C. 3& cycles of 30 seconds denatoration at QB^C, 30 seconds annealing at optimal temperatore. and 30 seconds extension at 72°C and 3 min final extension at 72°C was used in ttie Eppendorf PCR system. Next. 10 pi of PCR product was analyzed on 2% ethklium bromide-containing agarose gel at 70 V for 20 min. The result was obsen/ed under UV light (GelDoc-lt 310, UVP) and compared virith ladder (EZ LoadTM. lOObp).
RESULTS
Producing Infected samples
Inoculating samples with A. parasiticus, tiie result showed that tiie fongus exposed on surface of peanut kemels aftarS-S 1 0.5 days and on surface of com kemels after 4.4 ± 0.5 days. TTie samples were already identified as infected samples (Figure 1). However, to select samples for PCR detection, we intend to choose samples when they have not exposed Ifie, infection outside yet Tbus. the samples for forttier DNA extraction were collected at tiie second day vwth peanut and al, the third day in case of com.
Figure 1: Appearance of A parasiticus on Infected com and peanut kamels. (a) Com kemels were inoculated vAlti A. paras^lfc for 2 days; (b) Com kemels were inoculated with A. parasiticus tot 4 days, (c) Peanut kemels were inoculated wflh A. parasflfcus h ^ days; (d) Peanut kernels were Kioculated with A parasiticus 3 days. A. parasiticus becane visible in the 3"* days witti peanut »d riPs 4 with com kemels. ' f^ >
Fungal collection and DNA extraction
After ttie infection pnDcess, fungal DNA was randomly exti-acted fnjm kemels, vrfiich was collected from tiie previo|, prepared samples ttirough direct mettiod and indirect mettiod After the exbaction. ttie concentration of DNA s a m *
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was measured to evaluate two methods.
Direct method .
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F^ure 2: DMA concentration isolated from dHferant number Rgure 3: DNA concentration Isolated from different number of Infected com kemels in direct method. of infected peanut kemels in direct method
DNA was extiacted from fungi grown on surface of kemds In ttiree sub-samples 3, 5 or 10 kemels. Tbe non-infected samples vrere used as negative conttols. In b^eattnent group, figure 2 shows the DNA concenttatton Increases (2.54 ± 0.74, 8.76 ± 3.08. 9.34 ± 1.83 ng/ul), respectively wrth ttia amount of kemels. Moreover, ttie kemels from ttne confrol group also gwe us DNA with ttie concenti-ation 1.52 ± 0.37 ng/ul (3 kemels). 2.87 ± 0.82 ng/ul (5 kemels), and 2.97 ± 0.75 ng/ul (10 kemels). Figure 3 also reveals tiie DNA coneenttations of beattnent group (6.4 ± 2.02 ng/ul to 14.09 ± 3.18 ng/ul) and confrol group (3.07 ±0.82, 3.57 ± 0.64 and 6.89 ± 1.47 ng/ul). tn comparison bebreen conbtil group and freatinent group, ttie DNA concenbBtion of the first group is lower than ttie latter.
indirect method
In ttie indirect method, fongal DNA was extracted from three and five kemels of peanut and com. which had been incubated in malt extract agar for 24 hours to enrich the fongi on ttie surface of ttiese kemels.
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Figure 4: DNA concentration Isolated from different number Rgure 5: DNA concentration Isolated from di^rent number of infected com kemels after enrichment of Infected peanut kamels after enrichment
In indirect method, the quantity of DNA was very high in comparison with direct mettiod. Figure 4 reveals that the DNA concentration from enriched samples are around 45-60 ng/pl in infected com kemels and about 14 ng/pl in controls group. The DNA concentration from 5 kemels is higher tiian tiiat from 3 kemels. Moreover, for exttaction form peanut kemels. the concentration of DNA in the treatments are around 156-168 ng/pl and 25-37 ng/pl in control samples (Figure 5). The DNA concenfration from freatinent group is also much higher ttian one from confrol group.
The extracted solution fram freatment gmup might contain DNA of the target fongus and other sources from the substrate. That was ttie reason why the quanttty of extracted DNA in freatment group was higher ttian ttie one In contixil group for botti cases, direct and indirect methods, tt suggested friat ttie extiacted DNA in treatments can be used in order to stody whether host' DNA affects on PCR in the next stage or not. In addition, frie DNA concenbation in indirect metiiod was at least 5 times higher than tiie one in direct method because of the fongal enrichment process.
Detection of A. parasiticus by PCR assay 0/recf/net/iorf
Based on the lowest concenfration (about 3 ng/pl). for each PCR reaction. Uie minimum volume of exfraeted DNA was 4 pi used for A.parasiticus' detection. It was also utilized to analyze the specificity and to estimate the sensitivity of the assay when the DNA samples were contaminated by host and other" DNA because, in reality, the extiacted sample will contain other DNA sources rather than fongal DNA and tha target fongal DNA cannot be quantified by conventional PCR.
In figure 6. PCR products of all six lanes appear. All bands are clear and bright except the band ftom lane 2 corresponding to lowest DNA eoncenttation from three com kemels. This blurrlest band was selected for limit of
riPfPrtion ( L O D I T h e bands' bright intensities increase according to ttie increase ot D N A concenfration. In addieon;ig n e g X V c o n f r o l lane did not appear a n y band This result proved that ttiere w a s no terget D N A in negative control.
Fiaure 6" PCR result from infected kemels in direct mettiod. Figure 7: PCR result frx>m infected kemels In indirect Extract
^ ^ DNA from 3. 5, 10 infected c o m kemels (lane 2-4); DNA from 3, 5 Infected c o m kemels (lane 2, 3); Exfrwt from Extracted DNA from 3, 5, 10 infected peanut kemels (lane 5- 3, 5 Infected peanut kemels (lane 4, 5) and (lane G, 7) negitlve 10) and (lane 8) negative control (non-infected samples). controls (non-infected samples).
Indirect method
Higher DNA concentiation was obteined after ennchment process, thus e a c h P C R assay w a s used 2 pi of DNA template exfraeted from infected peanut and c o m kemels. The negative controls w e r e e x t i a c t e d from five kemels of com and' peanut wittiout A. parasiticus Inoculation. Figure 7 shows four bright b a n d s u n d e r UV photometer. In addltitm, two b i ^ of negative treafrnents do not s h o w a n y band. It means that ttie samples w e r e not c o n t a m i n a t e d by A. parasiticus. ,^ ^ For both direct and indirect mettiod, in all samples, there Is only o n e 3 4 3 b p b a n d in P C R product, no smear o ^ ^ bands are observed. It is knovm tiiat ttie extracted D N A solution c a n contain o t h e r D N A sources beside our ^ S genome, but ttiere is only o u r terget product show up. T h u s , this pair of p n m e r s is highly specific for A. patasiUm
detection. '
DISCUSSION E Foods and feeds are espeaally susceptible to the colonization of aflatoxigenic Aspergillus species in tropical areas'
during pre-harvest. processing, tiansportatlon, and storage. T h e fungal quantification a n d qualification are importmfl elements to contit)! the raw matenal quality and to predict ttie potential risk of afiatoxin presence (Shapira et ai. 1996).' In ttiis stody, an A. parasiticus-deteeing pnacedure was studied to archive a n e w a p p r o a c h toward this problem. FortJ^
purpose, ttiere were h r o groups of freattnents earned out on artificially Infected c o m a n d peanut kemels to provide fungal
DNA for PCR assay. J In the first step of the study, artificially Infected samples w e r e produced. It is easily to Infect peanut and com kanels in
short time, about 2 to 4 days, and these kemels can be considered as infected f o o d . This point c o n f i m i s that ttie fongi wll|
grow u p very fast under suitable condition (wann and moist enough). Besides that, the Infection time of peanut and com is also difTerent. It Is twice for c o m in comparison with peanut. That may b e d u e t o t h e pericarp covering the com kernels^
which absents from the peanut kemels. This makes the fongi cannot reach the nutiient source easily. Among agncuttura*
commodities, peanuts are found to have the highest incidence of afiatoxins ( S t i a n g e , 1991) This point quite matches',
witti our sumiise. i In this wori<, the detection of A. parasiticus on artificially infected dried f o o d using P C R m e t h o d was canied out,
pemnit to predict ttie presence of a f l a t o n n s . To achieve this goal, t w o infected s a m p l e preparation processes were I to obtain the suitable o n e for DNA collection In direct mettiod. the obtained D N A concenfration w a s 3 to 10 ng/pnor-j c o m and 6-14 ng/pl for peanut w h e r e a s . In indirect procedure, those are a r o u n d 50- 6 0 ng/pl for infected com a ^ l ^ ' 170 ng/pl far peanut. It is reasonable because Uiere are m u c h more fungal cells growing on the suri'ace of kemW^alle^
e n r i d i m e n t process. Generally, in botii cases, D N A quantity w a s disordered because Infected kemels were ifndqmly collected. In control group, although tiie samples were not inoculated with A. parasiticus, D N A quantity still eidsts. Ci^
means tiiat ttiese DNA samples were contaminated with other D N A sources. T h i s w e a k n e s s of the experimental deslgc can still help us to prove tiie specificity of primers. Provided that D N A s a m p l e s w e r e contaminated and tiie primer canj
only amplify the terget product, the pnmer pair is specific. , In addition, the result ftom PCR assay brings us a conclusion about the efficiency o f t w o treatinent methods: d l f ^ anQ
indirect mettiod. The direct method has potential to provide us good e n o u g h D N A samples for PCR reaction. T o ^ t t for this idea, DNA eoneenfratton in indirect method is about 10 times higher than t h e o n e in direct method. H o w e v e r ^ indirect mettiod spends about 2-3 d a y s more far fungal enrichment process. Moreover, the direct method can helpii eliminate ttie contemination occurring during the enrichment process. In this m e t h o d , D N A quantity and quality areatei satisfying writh good detection in short time. Besides that, ttie sensitivity of P C R reaction, in case D N A samples i r i ^ i contain other DNA sources rattier than the target DNA, was provided by ttie limitation of detection (LOD) coming f f * ! the bluned band of ttiree c o m kemels, LOD = 12ng. In previous study, the sensrtivity of P C R reaction was d e t e m i i i ^ J i the concentiation gradient of D N A exttacted from A. parasiticus' pure cultore. A n estimated LOD of target DMA, i<^Mi case, w a s 0.005 ng/ul. This result could be acceptable in comparison witii L O D 0.0025 ng/ul in another study nf^
realtime PCR mettiod (Sardinas ef al., 2011). In addition, LOD of P C R reaction using D N A extracted fnam arffl"
infected kemels is about 240 times higher than the one using D N A extracted from p u r e fungal culhjre. It sugges' PCR reactions to detect A. parasiticus on infected samples need more D N A a m o u n t d u e to the contamination o' Dr^lA sources such as from host and ottier microorganism on the kemels.
In addition to hearth c o n c e m related to aflatoxin, ttie rejection o f eonteminated peanut a n d c o m . w h i c h contain unacceptable a m o u n t o f aflatoxin. results in large e c o n o m i c kJss. For most species, ttie letiial d o s e (LD50) ranges ftxjm 0.5 to 10 mg/kg b o d y w e i g h t T h e r e f o r e , ttie success o f Biis stody will contribute t o i m p n w e f o o d safety and to avoid the c o n s u m p t i o n o f c o n t a m i n a t e d food.
C O N C L U S I O N
In this study, w e c o n c e n t r a t e d o n developing a protocol t o detect A. parasiticus b y P C R a s s a y using D N A isolated f r o m Infected s a m p l e s . T h e direct D N A isolation w a s suitable f o r ttie detection o f A. para^icus o n infected f o o d a n d feed b y ttie d e s i g n e d P C R a s s a y . T h e r e c o m m e n d e d D N A a m o u n t f o r P C R reaction is at least 12 n g . It comes fram 2 g o f infected f o o d . W r t h t h e direct m e t h o d . A. parasiticus could b e detected within two d a y s . It i s a rapid a n d sensitivity protocol, w h i c h provides a usefol t o o l for eariy detection o f A parasfficus in dried f o o d and In other food s y s t e m s . It i s r e c o m m e n d e d t h a t this d e s i g n s h o u l d b e applied to identify ttie fungus in natorally infected food a n d f e e d .
A C K N O W P i L E D G E M E N T
This research isfimdedby Vietnam National University - HoChiMinh City under grant numl>erB2013-28-02.
R E F E R E N C E S
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on microbiological specifications for food. Toxigenic fungi' Aspergillus. Microorganism in foods
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Romwlab (2012). Mycotoxins. Renter Labs guide to mycotoxins (4th EdSon).
Sardinas N, Vazquez C. Gll-Sema J. Gonzalez-Jaen MT and Patino B (2011). Specific detection and quantification of Aspergillus flavus and Aspergillus parasiticus In wheat flour by SYBR(R) Green quantitative PCR. Int J Food Microbid 145 (1): 121-125.
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Mycotoxins and animal foods. CRC Press, Boca Rat, FL 341-362.
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P H A T H I E N N A M M O C APERGILLUS PARASITICUS TR^N N G O V A D A U PHONG B! N H I E M T H U D Q N G
H u ^ h L g T h a o T r i n h , T h a n T h j M y L i n h , N g u y i n T h j H u i *
Tnrdng 0 9 / Hgc Qu6c Ti, Bd Hoc Qudc Gia Tp. Hd Chi Minh '
T 6 M TAT
Thvrc pham v i thiic fin chlin nii6i bi nhilm d^c t6 tur cac loii nim Aspergillus dugc xem nhir mOt vln d^ c6 koh hirdmg ldn d|n s(rc khde con nguoi v^ v|it nu6i. Aflatoxin Ik m§t trong nhung dpc 16 nam moc dang chfi y nhSt va dugc biet dSn nhu cic chat chuyen h6a sSy ung thir Aspergillus parasiticus li ic§t trong nhung loai sinh ra dpc t6 nay. Hi?n nay, viec phdt hiSn loii nim niy trfin diuc phim milt a l nhiiu thW gian do svr giSng nhau v^ hinh ttiii giOa cic loii n k i . Do do, cd nhi^u nghien cmi vc cfc
nim ni6i d^ loai b6 nhOng ttivrc phim nhiSm dgc. N ^ e n cihi niy da ^ dung phirong phip PCR de phSt hipn A. parasiticus tren nhfing mlu thirc phim bj nhiSm thvi dOng. A. parasiticus dirge cho nhiem trSn cac mau h^t d di6u kifn gan gidng yin dieu ld?n nhiSm trong tvr nhienl ADN ciia nim trong phin ilng PCR thu dupc bing hai cich - true tiep tren mau nhiem vi giin tilp sau qui trinh lim (Tiiu nim Kit qua li vi^c phit hiSn A. parasiticus da diinh cong tren ci hai loai mau trgn bing phirong phip PCR. Phin ung PCR cin It nhit 12 ng ADN trich dupc tu 2 g ttivrc phim nhilm nim. So sinh hai phuong phip trfin. ADN trich trvrc tifip ^ mlu nhiem nim thich hpp cho PCR hon^ Do d6 phuong phip niy dupc de xuat nhu m6t phirong phip moi nham phit h i ^ A parasiticus nham trinh sir dving tb\fc phim nhiem dpc.
Tir khda- /jflatoxins. Aspergillus parasiticus, h?t b ^ , phuong p h ^ tryc t i ^ , phumig phip giin tifip, hat diu, PCR.
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