A Comparison of Four Molecular Methods for Detection of Aflatoxin-Producing Aspergillus in Peanut and Dried Shrimp Samples Collected from Local Markets around Pathum Thani Province, Thailand

Aspergillus flavus is an aflatoxin-producing fungus which is poisonous to humans and animals when consumed. Detecting the fungus can help to prevent this danger. The four molecular methods, namely, conventional isothermal amplification (LAMP), PCR, quantitative LAMP (qLAMP), and qPCR, were compared to determine their efficiency for A. flavus detection. Thirty samples of peanut and dried shrimp were collected from 15 markets around Pathum Thani Province in Thailand. The samples were artificially infected with 108 conidia/ml of A. flavus for 1 hr and enriched for one day to represent real contamination. The results show that the sensitivity detection for A. flavus in PCR, LAMP, qPCR, and qLAMP was 50 ng, 5 ng, 5 pg, and 5 pg, respectively. Aspergillus in 30 peanut and dried shrimp from the market was detected by all four methods. The detection rate was about 20%, 60%, 100%, and 100% with PCR, LAMP, qPCR, and qLAMP, respectively. The molecular detection technique, especially LAMP, qPCR, and qLAMP, can detect this pathogenic fungi very rapidly with high sensitivity and reliability in comparison to conventional PCR.


Introduction
Food safety problem is the important problem that is a threat to the health of the population [1,2], especially in the developing countries. e major trouble of food safety is food poisoning by the contamination of microorganisms such as bacteria and fungi. is study emphasizes only the fungus, particularly genus Aspergillus. e genus Aspergillus includes more than 600 species; about 40 species are known to cause disease in humans [3].
is dangerous species produces aflatoxins. Aflatoxins are a group of mycotoxins that display acute immune and hepatotoxicity in humans. Aflatoxins also exhibit very high carcinogenic potential following chronic exposure [4,5]. Aspergillus section Flavi includes species capable of producing a wide array of toxins among which aflatoxins are the most important for food safety. Aflatoxigenic fungi have contaminated many kinds of food and cereal [6,7] such as dried fruits, nuts, edible seeds [8], rice, spices [9], and herbs [4,10]. Food and feed are particularly susceptible to colonization by aflatoxigenic Aspergillus in warm and dry weather. Aflatoxigenic fungi can produce toxin at several stages of the food chain, such as preharvest, processing, transportation, and storage [11,12]. e aflatoxigenic fungi species is divided into three sections of the fungal genus Aspergillus, i.e., sections Flavi, Nidulantes, and Ochraceorosei [13]. Among these, section Flavi includes 16 species capable of producing a wide array of toxic compounds [14]. However, A. flavus and A. parasiticus are significant because they produce aflatoxin in food [15]. A. flavus and A. parasiticus have different toxicogenic profiles. A. flavus produces aflatoxin B1, B2, cyclopiazonic acid, aflatrem, 3-nitropropinic acid, sterigmatocystin, versicolorin A, and aspertoxin, whereas A. parasiticus produces aflatoxin B1, B2, G1, G2, and versicolorin A [16].
In this study, the Aspergillus norsolorinic acid reductase gene (NOR1) was selected for molecular detection with PCR, qPCR, LAMP, and qLAMP. e gene is involved in aflatoxin B synthesis. e nor1 encodes norsolorinic acid reductase.
is enzyme converts norsolorinic acid to averantin. However, researchers have investigated other genes involved in the biosynthesis of aflatoxins. e multiplex PCR technique with three genes, aflM, aflP, and aflR, was used to detect Aspergillus flavus in peanut, corn, and three insect species commonly found in stored grains [14]. Reddy et al. [15] used aflR and aflO to detect A. flavus contamination in rice samples. However, not only genes were used in the aflatoxin biosynthesis to determine the Aspergillus contamination.
erefore, efficient methods of identifying and detecting aflatoxigenic fungi in food and raw ingredients should be developed. Currently, molecular methods such as polymerase chain reaction (PCR), real-time PCR or quantitative PCR (qPCR), and loop-mediated isothermal amplification (LAMP) are effective for detecting food contamination by microorganisms.
Nowadays, LAMP has become the interesting method to replace PCR due to its more rapid, simple, and specificity reaction. In this present time, LAMP has been developed and used in many applications, such as for sex determination in human [17], detection of pork meat in Halal food [18], detection of Yersinia enterocolitica in pork meat [19], and detection of Salmonella infection in chicken samples [20] is method was performed under isothermal conditions in the temperature range 60°C-65°C for 60 minutes [21]. ere are two sets of primer; inner primer and outer primer sets used in LAMP were specific at six different DNA sequences within the target DNA, and primary DNA amplification was begun by the inner primer set. e characteristic intermediary DNA structure formed by LAMP, called a stem-loop DNA fragment, was generated, and large amounts of DNA products were produced by an autocycle reaction [22]. en, this research aims to compare the efficiency of the PCR, qPCR, LAMP, and qLAMP methods for detecting aflatoxin-producing species within Aspergillus section Flavi and to apply these methods to detecting fungi purified DNA, spore suspension artificially contaminated samples, and naturally contaminated peanut and dried shrimp collected from local markets around Pathum ani province, ailand. e agar plates were incubated at 28°C ± 2°C for 7-10 days and then monitored daily for the appearance of fungal colonies. e DNA was extracted from the isolated fungi using a fungal DNA extraction kit (PrestoTM Mini gDNA Yeast kit; Geneaid, New Taipei City, Taiwan), and the DNA concentration was measured using a NanoDrop 2000c Spectophotometer ( ermo Scientific).
e nucleotide sequences of each primer are shown in Table 1.

PCR and qPCR Reactions.
A PCR assay targeting the Norsolorinic acid reductase gene was performed in parallel with the LAMP primers to compare the detection efficiency, as shown in Table 1. SYBR green I dye was used to enhance the specificity in the qPCR reaction. e PCR amplification contained 1 × Taq DNA polymerase buffer, 1.2 mM dNTPs, 0.8 μM F3 and B3 primers, 8 U Taq DNA polymerase (New England Biolabs), and 5 ng of each DNA extract as a template in a final volume of 25 μl. e cycling conditions comprised of a single initial denaturation at 95°C for 3 min followed by 35 cycles at 90°C for 30 sec (denaturation), 56°C for 30 sec (annealing), 72°C for 30 sec (extension), and a final extension step at 72°C for 5 min. After the PCR amplification, the products of 550 bp were analyzed by electrophoresis using 1.5% agarose gel and were analyzed by Gel Doc TM XR+ with Image Lab TM Software (BIO RAD, USA).

LAMP and qLAMP Reaction.
All LAMP reaction were performed followed the methods of Kanchanaphum and Vichaibun [20] containing 5 mM MgSO 4 , 400 mM betaine (Sigma), 1.2 mM dNTPs, 0.8 μM F3 and B3 primers, 2 μM FIP and BIP primers, and 8 U Bst DNA polymerase (New England Biolabs). After the LAMP reaction, the products were analyzed by electrophoresis using 1.5% agarose gel and were analyzed by Gel Doc TM XR+ with Image Lab TM Software (BIO RAD, USA.). e qLAMP amplification was performed by adding 0.5 μL of SYBR green I dye (Invitrogen, Carlsbad, CA) to the normal LAMP reaction.  [23], and Siruguri et al. [24] were followed. en, all samples were dried in a laminar hood under ultraviolet light for 3 min. After that, the peanut and dried shrimp were inoculated with 10 8 conidia/ml of A. flavus. e samples were incubated for 1, 3, 6, and 24 hrs., respectively. en, the infected samples were cultured in PDA for 1 and 2 day for enrichment time. After that, fungi DNA extraction was performed.

e Detection of A. flavus in Peanut and Dried Shrimp Samples Gathered from Local Markets.
irty peanut samples and thirty dried shrimp samples were collected from 15 local markets in Pathumthani Province, ailand. After the purchase, all of the samples were stored in an ice box and immediately delivered to a laboratory for DNA extraction and further experiments.

e Specificity Test of the PCR and LAMP.
e results of the specificity tests for the PCR and LAMP methods are shown in Figures 1(a) and 1(b), respectively. Both methods demonstrated 100% specificity when tested with non-Aspergillus flavus strains.

e Sensitivity Test of the PCR, qPCR, LAMP, and qLAMP.
Ten-fold dilutions of the DNA template were used to compare and determine the detection limits of all four methods. For the PCR assay, 550 bp PCR product was found in 1 and 2, as shown in Figure 2(a). e detection limit for the PCR assay was 50 ng. e detection limit for the LAMP reaction was 5 ng, as shown in Figure 2(b). erefore, the sensitivity for both qPCR and qLAMP was 5 pg, as shown in Figure 2(c) and 2(d). e R 2 value from standard curve of cycle threshold from qPCR was 0.9996, and the R 2 value from standard curve of time to detection from qLAMP was 0.9292, as shown in Figure 3.
In Figure 2 So, the sensitivity of each method (PCR, qPCR, LAMP, and qLAMP) was summarized in Table 2.

e Detection of A. flavus in Spiked and Naturally Contaminated Peanut and Dried Shrimp Samples from the
Markets. For artificial infection, the samples were incubated for 1 hr and then cultured in PDA for 1 day. e samples were incubated for 1, 3, 6, and 24 hrs., respectively. en, the infected samples were cultured in PDA for 1 and 2 days for enrichment time. After that, fungi DNA extraction was performed.
A total of 30 peanut samples and 30 dried shrimp samples collected from 15 markets around Pathum ani province, ailand, were enriched for one day. After DNA extraction and amplification with all four assays, the results were obtained, as shown in Table 3.

Discussion
In this study, the Aspergillus norsolorinic acid reductase gene (NOR1) is selected for molecular detection with PCR, qPCR, LAMP, and qLAMP because it is highly effective at detecting the A. flavus contamination in food.
ese primers showed high specificity and sensitivity. Beyond the NOR1, the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) were also used to detect the Aspergillus. Levin [25] reported that the sensitivity of ITS1 and ITS2 PCR primer for A. parasiticus CBS 126.62 was 9.03 pg, while our result for the PCR detection using the nor1 gene target was 500 ng. Our sensitivity was quite low compared with Levin and colleagues [25]. So, the PCR condition such as the concentration of magnesium ion may be adjusted for the better sensitivity. e sensitivity of PCR primarily depends on three factors: the physicochemical condition of the reaction, the concentration of DNA template, and the specific primer [26]. However, our results for the LAMP reaction was 50 ng, which was quite similar to the findings of Luo et al. who used acl1 (ATP citrate lyase subunit 1) to determine the contamination [27]. eir sensitivity result for the LAMP reaction was 20 ng [27], which implies that the sensitivity of the LAMP method is better than the PCR method. qPCR and qLAMP have been used to enhance performance. Freckle et al. [28] used the qPCR method to detect and characterized the Aspergillus species. Schabenreiter-Gurtner et al. [16] also used qPCR to detect the different Aspergillus and Candida species by targeting the ITS-2 region of the fungi. Both Frickle S. et al. [5] and Schabenreiter-  -3′  F3-nor1  ACT GCG ACT CGG AAA GCG A  B3-nor1  GGA CTG CTG CAG CAT CAG  FIP-nor1  GGC CCA AAG TTC TGC GCC AT-C CAG ACA TTG CGG GAG GA  BIP-nor1 ACC ATG CCC CTC GAG GAT CT-G CGG GTT GCC TGA AAC AG Nor1-F CTTTTCTCCAACGTCCCAAA Nor1-R ACAAGAACCCTCGGACTGTG Gurtner et al. [16] used hybridization probes to perform the qPCR detection, which was expensive and complicated. erefore, in this research, a method using SYBR green fluorescence was developed. is was more cost effective and less complicated than the hybridization probe method or the TaqMan method, which is based on dual labeled olignonucleotide. e results of this research demonstrated that the qPCR (50 pg) sensitivity detection was 10,000 times better than conventional PCR (500 ng) sensitivity detection. Like the qPCR method, the qLAMP method improved the efficiency of the conventional LAMP method. e performance of the qLAMP method (50 pg. of DNA) was 1,000 times better than the conventional LAMP method (50 ng. of DNA). ese results were according to Marmiroli and   Maestro [28]. ey reported that the limit of detection by ethidium bromide staining is about 5 ng DNA (visual inspection) and 60 pg DNA by SYBR Green I [29]. e limit of detection by ethidium bromide staining is about 5 ng DNA (visual inspection) and 60 pg DNA by SYBR Green I [29]. From this study, it can be seen that the detection rate of Aspergillus infection in peanut and dried shrimp from the market were very low using PCR, 20% positive in peanut and 13.33% positive in dried shrimp (from Table 3). e main reason of high numbers of negative result is the limitation of PCR.
e efficiency of PCR technique is 50 ng DNA of pathogen, while the efficiency of LAMP and both qPCR and qLAMP are 10 and 10 4 times, respectively, higher than that of PCR. So, the LAMP has about 3 times proficiency than PCR, while qPCR and qLAMP have about 5-7.5 times proficiency than normal PCR (from Table 3). e analysis' capability to detect low amount of A. flavus in peanut and dried shrimp was confirmed. On the other hand, all four methods required the complex equipment, such as a gel-electrophoresis apparatus or real-time PCR machine, to detect the DNA amplification. Further research could develop the lateral flow dipstick or LFD method to detect the DNA amplification product because it is easier and less complicated. e LFD method can also detect the amplification product without a gel-electrophoresis apparatus, which is useful for field studies.

Conclusions
e results show that PCR, LAMP, qLAMP, and qPCR methods could detect Aspergillus contamination in peanut and dried shrimp. Especially, both LAMP and qLAMP methods were more rapid, reliable, and robust for Aspergillus detection in the samples and may be useful molecular tools for routine testing. In addition, qLAMP and qPCR methods were the most efficient in terms of sensitivity.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.