Advanced DNA-based methods for the detection of peanut allergens in processed food

Trends in Analytical Chemistry 114 (2019) 278e292

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Trends in Analytical Chemistry journal homepage: www.elsevier.com/locate/trac

Advanced DNA-based methods for the detection of peanut allergens in processed food Mengyao Zhang a, Ping Wu b, Jun Wu c, Jianfeng Ping a, Jian Wu a, d, * a

College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China Hangzhou Wahaha Group Co, LTD, Hangzhou, 310018, China c Lin'an Center for Disease Control and Prevention, Lin'an, 311300, China d Key Laboratory of on Site Processing Equipment for Agricultural Products, Ministry of Agriculture, Hangzhou, 310058, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 5 February 2019

Currently, food allergy is a worldwide public health problem, of which peanut allergy is more severe. Peanut allergy is usually lifelong and even trace amount of peanut allergens can cause severe anaphylactic reactions. Nowadays there is no complete cure for sensitized individuals. The dominant prevention is to avoid exposure to peanuts and peanut products. But there are risks of false ingredient labeling and cross-contamination during food processing. Hence, it is important and urgent to detect peanut allergens in food products. This review highlights advances and future trends in DNA-based methods applied to detect peanut allergens in processed food. A summary of published methods for detecting peanut allergens in food is given with a comparison of DNA targets and limit of detection. © 2019 Elsevier B.V. All rights reserved.

Keywords: Allergy Peanut allergens Polymerase chain reaction Isothermal amplification DNA hybridization

1. Introduction Currently, food allergy has grown more and more concern around the world, which is defined as an overactive immune response causing by a given food [1e3]. According to Food and Agriculture Organization (FAO), the most common allergenic foods include milk, egg, fish, crustaceans, peanut, soybean, tree nuts and wheat accounting for 90% food anaphylactic reactions [4]. Among them, anaphylactic reactions to peanut is more adverse. Even trace amount of peanut can induce severe anaphylactic reactions related to cutaneous, gastrointestinal or respiratory manifestations with a possibility of death for highly sensitive individuals [5]. The number of allergic individuals is about 1.5 million in USA and 50e100 people die from peanut allergen every year [6]. The prevalence of peanut allergy has been increasing in recent years [7]. Peanut allergy is usually lifelong and only 20% sensitive individuals can overgrow it [8]. Although studies have shown that immunotherapy can ease allergic individuals' reactions to peanuts or that gradual ingestion of foods containing trace amounts of peanut protein during childhood can improve their tolerance to peanuts and peanut products, these methods have not reached the level of

* Corresponding author. College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China. E-mail address: [email protected] (J. Wu). https://doi.org/10.1016/j.trac.2019.01.021 0165-9936/© 2019 Elsevier B.V. All rights reserved.

clinical treatment [9,10]. Thus, there is no complete cure for peanut allergy actually. The dominant prevention is to avoid exposure to peanuts and peanut products [11]. Nowadays peanuts are widely consumed in our daily life since they have delicious taste and rich nutritional and economic value. There are many commercial food products containing peanut ingredient or using the same production line with peanut on the market. These allergen messages will be indicated on the label of food products. However, the potential risk is the presence of hidden allergens due to crosscontaminations during food processing, which is quite dangerous for allergic individuals. Hence, it is of great necessary and urgent to detect peanut allergens in processed food to ensure the avoidance of exposure to peanut-containing food for allergic individuals. Peanut allergy represents a hypersensitivity reaction to peanut proteins. The detection methods are divided into two categories. One is to detect allergenic proteins directly and the other is to detect the genes encoding an allergenic protein or other specific DNA fragments [12,13]. Enzyme-linked immunosorbent assay (ELISA) is a common protein-based method that detects allergenic proteins based on the interaction of the species-specific proteins with antibodies and it is widely used by food industry and official food control agencies. There are several commercial ELISA test kits available for peanut allergens such as Veratox, BioKits, ELISA-TEK and Ridascreen [14]. The limit of detections (LOD) are in the range of 1e5 ppm. Although

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Abbreviations CTAB DoE DVD ELISA FAO GMO HAD HRP-SA IAC IgE ITS1 LAMP

cetyl trimethyl ammonium bromide Design of Experiments digital versatile disk enzyme-linked immunosorbent assay Food and Agriculture Organization genetically modified organisms helicase-dependent amplification streptavidin horseradish peroxidase internal amplification control Immunoglobulin E internal transcribed sequence 1 loop-mediated amplification

ELISE can be highly sensitive and direct, food processing methods such as heat treatment affects strongly on protein conformation and quality and the results may be misled by cross-reactivity with other nuts [15e18]. Mass spectrometry (MS) method is a nonimmunological protein-based method that detects the presence of allergens. This assay can be effective and accurate to discover allergens or epitope encrypted in protein moieties [19]. However, it does require pricy and sophisticated instruments, complex operation and data analysis, which is time-consuming and expensive. Since the large quantity of peanut products on the market, the trend of peanut allergen detection must be fast, simple and convenient. Therefore, the rapid, sensitive and simple DNA-based methods are an alternative assay to direct protein detection, in which some emerging methods can be run without large-scale equipment [20]. These methods include nucleic acid amplification-based [e.g. polymerase chain reaction (PCR), digital PCR, isothermal amplification] and DNA hybridization-based assay (e.g. electrochemical biosensor, optical biosensor). Although DNAbased methods are indirect detection approaches of peanut allergens based on a segment of the gene encoding allergenic protein, plenty of experiments have shown that it is a promising detection method with high sensitivity [6,21]. Another advantage is that DNA is more stable than protein during food processing and extraction as well [21]. Our review does intend to provide a full-scale overview about emerging DNA-based methods for peanut allergen detection. The DNA targets which is the emphasis of these methods will be discussed particularly. The DNA-based methods available for peanut allergen detection will be compared and the development prospect will be proposed. This may inspire further study on rapid detection methods of peanut allergens. 2. Sample preparation of DNA-based method DNA target is the emphasis and difficulty of DNA-based methods, which has a great effect on detection results. DNA target for these methods can be genes that encode an allergenic protein or any other specific DNA marker such as mitochondrial, chloroplast and other highly repetitive sequences. Screening for appropriate DNA fragments and good quality of extracted DNA might have a great effect on improving accuracy and sensitivity of DNA-based detection methods. 2.1. Screening for appropriate DNA detection fragments The most common DNA targets used in researches are the coding sequences of allergenic proteins. According to the official

LOD NaOH MCH MS NASBA NCBI PCR pI RCA RPA RT-PCR SDA SMART

279

limit of detections sodium hydroxide magnetic capture hybridization mass spectrometry; nucleic acid sequence-based amplification National Center for Biotechnology Information database polymerase chain reaction isoelectric point rolling circle amplification recombinase polymerase amplification Real-time PCR strand displacement amplification signal mediated amplification of RNA technology.

allergen nomenclature database (http://www.allergen.org/), seventeen peanut allergens are acknowledged which are Ara h 1, Ara h 2, Ara h 3/4, Ara h 5 to Ara h 17, respectively. As the sera of nearly 90% of allergic patients has obvious recognition effect on Ara h 1, Ara h 2, Ara h 3/4 and Ara h 6, they are identified as major peanut allergens [22,23]. At present, there are many researches on the study of major allergenic proteins and a lot of reports of detection methods have been published [24e27]. Some characterization of these allergens is listed in Table 1. Among them, Ara h 1 has the highest content in peanut total protein, followed by Ara h 2. Ara h 1 occurs in the form of trimeric complexes, which is stable and resistant to gastrointestinal tract digestion [28]. Shefcheck et al. confirmed that the allergenicity does not change even if the allergenic protein conformation has transformed [29], which may cause false negative results in protein-based methods. Ara h 2 is the most sensitive protein in peanut allergens. It is identified by the serum Immunoglobulin E (IgE) of more than 90% of allergic patients [30]. The structure of Ara h 2 allows it remaining stability and integrity under harsh treatment [26]. Due to the hypersensitivity of these major allergens, their encode genes are usually used as DNA targets in DNA-based methods. The oligonucleotide primers and probes, used in DNAbased methods, are mainly designed upon the Ara h 1 gene, Ara h  et al. designed two specific sets of 2 gene and Ara h 3 gene. Ren
cova primers based on Ara h 1 gene and hazelnut Cor a 1 gene to develop a multiplex PCR system [31]. They also used the universal plant primers of chloroplast gene to eliminate the potential inhibition of PCR reaction. The developed system was cheap, specific and sensitive with a LOD of 0.001% w/w (10 mg kg
1). Zhang et al. designed specific primer sets based on the DNA sequence of Ara h 1 together with a competitive internal amplification control (IAC) to improve the reliability of PCR results [32]. IAC is an artificial DNA sequence and it is designed by compound primer technique (Fig. 1). The PCR method with IAC can show false-negative results effectively due to pez-Calleja the competition between the target DNA and IAC. Lo et al. developed a real-time PCR system using DNA makers in the Ara h 2 gene and the Internal Transcribed Spacer (ITS) gene [33]. And the nuclear 18S rRNA gene sequences from various plant and animal species were used as a positive amplification control. Results found that the PCR system with ITS primers showed higher sensitivity than that with Ara h 2 primer pair. In 2009, the effect of heat treatment on the detection of peanut allergens was studied by three real-time PCR methods targeting DNA sequences coding for Ara h 3 [17]. Compared with ELISA kits, the results found that both ELISA and PCR method were effected by roasting and had a similar decline extend. The study showed that ELISA kits and PCR method could complement each other to confirm a positive result. Both two

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Table 1 Partial allergenic proteins from peanut. Allergen Isoallergen (UniProt or Genbank No.) Molecular mass (kDa) and theoretic pIa Prevalence of IgEb binding Cross-reactivity [23]

NCBI GenBankc a b c d

Ara h 1 Ara h 1.0101 (P43238)

Ara h 2 Ara h 2.0101 (Q6PSU2) Ara h 2.0201 (Q6PSU2) 16.6 kDa; pI 5.8

Monomer 63.5 kDa; pI 4.6 Occuers as trimer of 180 kDa 30e80% [76] 42e100% [77] Other legume and tree nut vicilins and Ara h 2 and Ara h 3 AF432231 [32]

Ara h 3/4 Ara h 3.0101 (O82580) Ara h 3.0201 (Q9SQH7) Monomer 60.0 kDa; pI 4.6 Occuers as hexamer of 360 kDa 16e57% [76]

Ara h 5 Ara h 5.0101 (Q9SQI9) 14.0 kDa; pI 4.6

Ara h 6 Ara h 6.0101 (Q647G9) 15.0 kDa; pI 5.5

3e24% of birch or grass 86e92% [77] pollen allergic patients Other legume and tree nut vicilins Other profilins e.g. Ara h 1, Ara h 2 and 2S albumins from almond Bet v 2 and Phl p12 Ara h 3 and Brazil nut, and Ara h 1, and Ara h1,Ara h 2 and Ara h 6 Ara h 3 and Ara h 6 AY007229[50] AF093541 [51] NRd EF609643.1 [63]

pI: isoelectric point. IgE: immunoglobulin E. NCBI: National Center for Biotechnology Information database. NR: not reported.

methods have disadvantages but DNA-based methods are more stable as the variability between different ELISA kits are higher than that between PCR methods. From the published articles, we learn that most research targeted on the gene of Ara h 2, and then of Ara h 1. Few articles were targeted on the gene of other peanut allergens. The reasons for this might be that Ara h 2 accounts for the largest amount of peanut protein and is the most sensitive allergenic protein. Once detecting the presence of Ara h 2 sequences, there is a high possibility of allergy for peanut. The details are presented in the next section. Except for species-specific gene sequence, DNA sequence with high copy number can also be used in high sensitive and efficient DNA-based methods. In the plant cell, mitochondrial DNA and chloroplast DNA are in high copy number so that they could be appropriate DNA targets for DNA-based methods. Recent researches have shown that mitochondrial DNA and chloroplast DNA are used as targets to achieve a low detection level [34e36].

Mitochondrion is a cell organelle that is used to generate energy. The mitochondrial genes such as 16S rRNA, matR and cytochrome C oxidase subunits 1 and 3 have been used as specific DNA markers because of their high copy number which can result in high sensitivity [37,38]. In 2018, a study established PCR primers and probes targeting mitochondrial DNA sequence, achieving quantitative detection of trace amount of peanut (Fig. 2) [36]. The bait8/Cbait8 systems described in this research showed increasing sensitivity compared with the cell nucleus detection systems analyzed in parallel. The function of chloroplast in plant cells is to process photosynthesis. The analysis on chloroplast DNA finds that it is a single circular molecule and the molecule size varies from species to species (107e218 kb). The sequence has high consistency allowing comparison and alignment to design highly specific primers and probes (Fig. 3). Commonly, several chloroplast genes are used for species identification, such as matK [34], rpl16, trnH-psbA and the trnL region of the chloroplast tRNA gene [6] etc. But as the purpose of DNA-based methods is to detect the presence of the allergenic proteins indirectly, the selection of DNA targets should be determined taking the analytical needs and approaches into overall consideration before detection. Caroline Puente-Lelievre et al. had proposed a protocol of identifying and selecting suitable chloroplast markers for PCR assay [35]. According to this, we extended the workflow from chloroplast genome to the entire peanut genome combined with the experience of our study group. Generally, the workflow to select suitable DNA markers for the DNA-based detection methods in the laboratory are presented in Fig. 4. By testing the specificity of the system iteratively, appropriate DNA primers and probes could be chosen and finally a wellperformed detection method could be established combined with detection technology. 2.2. Extraction of DNA

Fig. 1. Schematic of IAC construction. Reprinted from ref. 32 Copyright 2014 Elsevier Ltd.

Besides the DNA targets, the DNA isolation method also has a great effect on the performance of DNA-based methods used in allergen detection. Many researches showed that yield and purity of isolated DNA were strongly determined by the isolation methods as well as the composition of the food matrix [39e41]. Food samples contain fat, sugar, protein and so on which may inhibit reaction rate and decrease detection sensitivity [42]. An optimal DNA extraction method should be simple and rapid and it could minimize reaction inhibition factors. The classical DNA extraction method is Cetyl Trimethyl Ammonium Bromide (CTAB) method, which separates DNA from sugar and protein by forming a complex with DNA. Good quality

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Fig. 2. Schematic view of the mitochondrial detection system for the quantification of peanut allergens. The atp6 primers and probe were identified on counting 18,192 in PeanutDB (41) and amplified a 104 bp fragment between positions 1747 and 1851 (black double-headed arrow). The amplicon was located upstream of the coding regions of the two conserved mitochondrial genes: ATPase subunit 6, atp6, and NADH dehydrogenase subunit 6, nad6. Primer sequences are underlined and probe sequence is shaded in gray. Corresponding competitor sequence is shown below the probe region, and the mutations that were introduced are in bold lowercase letters. Reprinted from ref. 36 Open Access.

Fig. 3. Alignment of matK gene sequences Arachis hypogaea (peanut), Juglans regia (walnut), Pinus koraiensis (pine nut), Prunus dulcis (almond) using Clustal W2 program. Species-specific primer sets were indicated by arrows. Yellow for peanut. Reprinted from ref. 34 Copyright 2018 The Korean Society for Applied Biological Chemistry.

and purity DNA could be obtained through CTBA methods. But the operation of CTBA method is complex and time-consuming. What's more, it needs some poisonous organic reagents. Nowadays there are commercial DNA extraction kits available on the market. They are based on silica-based spin columns and they separate DNA by multiple centrifugation. Different samples choose different kinds of DNA extraction kits. Articles published show that commercial kit (SureFood Plant X, Congen, Berlin, Germany) [43], commercial Plant Mini kit (Qiagen, Hilden, Germany) [31], the genomic extraction kit (AxyPrep Multisource genomic DNA minipre kit, Axygen, Bioscience, CA, USA) [44] are used to extract peanut DNA. These methods are convenient and easy to operate, but usually a commercial kit is expensive. Magnetic capture hybridization (MCH) based on magnetic beads is a novel method that is used for DNA extraction [40]. A specific probe is linked to magnetic beads via biotin e streptavidin interaction and can capture target DNA. The magnetic beads which are combined with DNA move directionally in the applied magnetic field and gather so that DNA can be extracted without high speed centrifuge (Fig. 5). The advantage of this method is simple and it adds no inhibition to DNA. But the preparation of optimal magnetic beads may be difficult.

With the development of DNA-based methods, DNA extraction methods require rapid and simple operations. There is no universal DNA extraction method available for all food matrices. For peanut DNA, there is still room to explore rapid extraction methods. Wang et al. developed an extremely simplified sample preparation by using Sodium hydroxide (NaOH) solution [45]. Without purification, the crude cell lysate was used as template directly to detect genetically modified soybeans. The crude DNA extraction method was also used to detect phytopathogenic bacterium [46]. Thus, whether rapid extraction methods can be applied to extract peanut DNA needs to carry out a research and should take detection methods used into consideration. 3. DNA-based detection method 3.1. Nucleic acid amplification-based assay Nucleic acid amplification is the most common technology used in DNA-based detection methods. The principle of it is that the DNA template will be amplified to a large amount under the action of primers and polymerase. Then the amplified target fragments can be analyzed by gel electrophoresis or fluorescent detection

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Fig. 4. Workflow to identify and select suitable DNA markers for the DNA-based detection methods.

Fig. 5. The workflow for DNA isolation and purification based on magnetic beads (Resource: http://www.medicinalgenomics.com/sensativax-plantmicrobial-dna-purification-kit/).

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[17,33,43,50,51]. By screening for appropriate target DNA fragments and optimizing reaction conditions, the specificity and sensitivity of detection has been improved. Assessment of the methods reported already are summarized in Table 2. In 2011, a novel detection method based on single-tube nested real-time PCR was developed to improve the sensitivity by an order of magnitude and eliminate false-positive results [52]. It had two pairs of primers (outer and inner) in one reaction tube and applied different annealing temperatures to control the two rounds of PCR. The real-time fluorescence single was detected in the second round. The sensitivity of the new system was found superior comparing with the initial realtime PCR system. But the design of inner and outer primers and probe should obey the relative theory and the annealing temperature should be optimized, which is complex and difficult. Based on threshold-calibrated competitive real-time PCR, Holzhauser et al. achieved the quantification of allergenic peanut in food (Fig. 6) [53]. This method allowed quantification of 10e1000 mg/kg peanut in various food matrices and no external standards were necessary by using the calibrated chocolate as a matrix. Multiplex PCR or Multiplex real-time PCR is an assay to detect multiple allergenic ingredients simultaneously, which is efficient in commercial food detection. Many researchers developed several cheap and sensitive multiplex PCR methods to detect peanut

technology. Nucleic acid amplification technology has advanced either in principle or in operation method recent years [47,48]. In this section, we view those published nucleic acid amplification methods for the detection of peanut allergens. And the emerging methods are emphasized here. 3.1.1. PCR/real-time PCR-based method PCR is the earliest nucleic acid amplification technology. It is widely used in detection fields since its high sensitivity, good specificity and repeatability. For peanut allergen detection, the most used DNA-based method is PCR. The amplification process mainly consists of three steps: high temperature denaturation (95 C), low temperature annealing (55e60 C) and medium temperature extension (72 C) [49]. The advantage of PCR is that trace amount of target DNA fragments can be amplified by millions of times since the newly synthesized DNA double strands are used as templates for the following circle of reaction. PCR products are usually detected by agarose gel electrophoresis. Real-time PCR is based on PCR technology and it can detect the amplification reaction real time by adding fluorescence substance. The accumulation of fluorescence signal can reflect the amplification of target DNA. Several real-time PCR methods were developed to detect hidden peanut allergens in processed food

Table 2 Published nucleic acid amplification-based methods for detection of peanut allergens. Methodology RT-PCR

Target gene

Amplicon size, bp

purification method

Limit of Detection/ Sensitivity

unifying the units

Food matrices tested

Ref

Peanut, sorghum, corn, barley, wheat, buckwheat, oat, rice, millet, soybean, peas, sesame, celery, commercial foods Raw and roasted peanut, nuts, oat, rice, wheat, commercial brands of food Peanut, cereals, nuts, legumes, commercial foods Peanut in pastry, sausage, chocolate or cake Raw and roasted peanut, nuts, wheat, rice, oat, millet, beans, commercial foods Raw and roasted peanut, commercial foods

[32]

Arah1 with IAC

389

CTAB

0.005% (w/w)

50 ppm

Arah2; ITS

125; 90

SDS

10 ppm in wheat flour; 0.1 ppm in wheat flour

10 ppm; 0.1 ppm

Arah2

86

<10 ppm

<10 ppm

Arah2

66

2 ppm in biscuit

2 ppm

Arah3

78, 105, 114

commercial kit (SureFood Plant X) Six commercial kits; CTAB Five commercial kits; CTAB

10 mg kg
1 in cookies

10 ppm

qRT-PCR

matK

75

0.001 ng

/

100; 69; 68 66; 105; 104

0.1e1 ppm

0.1e1 ppm

Peanut, nuts, soy, sesame

[35]

CTAB

10 ppm; 10 ppm; 1 ppm in milk powder

10 ppm; 10 ppm; 1 ppm

Peanut, soy, commercial foods

[36]

Multiplex RT-PCR

matK; rpl16; trnH-psbA Arah2; Arah3; ATPase subunits 6 Arah1

I-genomic Plant DNA Extraction Mini Kit; CTAB DNeasy Plant Mini Kit; SDS

180

10 mg kg
1 in wheat flour

10 ppm

66

20 ppm

20 ppm

Arah2

66

50 mg/kg

50 ppm

Arah2

194

Wizard Plus Minipreps DNA purification system Plant Genomic DNA isolation Kit

Peanut, hazelnut, nuts, wheat, beans, rice, commercial foods Peanut, cashew, hazelnut, celery, soy, mustard, rice cookies, tree nuts, commercial foods Peanut, hazelnut, celery, soy, egg, milk, almond and sesame

[31]

Arah2

Commercial Plant Mini kit Wizard Plus Minipreps DNA purification system

0.005% (w/w) in maize powder

50 ppm

Arah1; Arah2 Arah1;

54, 86; 54, 66 /

60 ppm, 60 ppm 60 ppm, 30 ppm /

ITS1 Arah6

/

0.006%, 0.006%; 0.006%, 0.003% 100 pg of peanut genomic DNA; 1 pg 0.4 ng/mL.

Digital PCR LAMP

LAMP microfuidic chip

CTAB AxyPrep Multisource genomic DNA minipre kit CTAB

0.4 ppm

Hazelnut, pistachio, oat, sesame, peanut, cashew, barley, wheat, soybean and pecan Raw and roasted peanut, Soybean, commercial foods Raw and roasted peanut, nuts, commercial foods Peanut, sesame, soybean, commercial foods

[33]

[43] [50] [51]

[34]

[54]

[55]

[58]

[60] [44]

[63]

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Fig. 6. Principle of competitive real-time PCR for threshold concentration-based quantification of peanut. Reprinted from ref. 53 Copyright 2014 Elsevier Ltd.

allergens in processed food [31,54,55]. However, multiplex PCR may be interfered with multiple food matrices and oligonucleotide primer pairs in one reaction, leading to low amplification efficiency [56,57]. To improve detection sensitivity, Cheng et al. developed a multiplex PCR assay combined with capillary electrophoresis to detect simultaneously 10 common food allergens including peanut [58]. Capillary electrophoresis has high resolution and could separate amplified DNA fragments rapidly and efficiently. This method was confirmed to have good stability and sensitivity by testing in three other laboratories. Since commercial food commonly contain more than one food allergen, multiplex PCR method is time and cost efficient. However, the detection sensitivity still has room to improve comparing with that in simplex PCR amplification.

3.1.2. Digital PCR-based method Digital PCR is a novel technology that combines the advantage of real-time PCR with a partitioned counting. It is based on fractioned PCR by dispersal of reaction volume in many droplets or cavity as separate reactions and the absolute concentration of the target copies in the initial sample is determined by counting the number of positive and negative partitions after end-point PCR amplification through the application of Poisson statistics [59]. So it could achieve accurate detection even in trace samples and complex samples. As far as we know, there is only one research published on food allergen detection based on digital PCR. In 2018, Pierboni et al. established a droplet digital PCR method to detect peanut and soybean allergens [60]. By evaluating different published real-time PCR methods, the research selected appropriate target sequences

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and the reaction conditions. Accepted numbers of droplets were 10,000 per reaction and mean copies per droplet did not exceed 0.4 molecules. Positive droplets that contained the target products were discriminated from negative droplets by applying a threshold set manually. This digital PCR system showed the same or a better sensitivity of real-time PCR and could discriminate the non-specific amplification. But the article did not present the results of the fluorescence signal of peanut system. What make sense is that this paper evaluated the applicability of digital PCR to detect and quantify the amount of allergens. It showed the possibility to develop a multi-screening analysis for detecting allergens in food in the future to overcome the inhibition of multiplex PCR. 3.1.3. Isothermal amplification-based method PCR requires thermal cycling procedures and commonly the whole amplification procedure takes 1e2 h, which is not suitable for rapid detection. In recent years, several isothermal amplification methods have been proposed to overcome the limitation of PCRbased methods, including nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), loopmediated amplification (LAMP), rolling circle amplification (RCA), signal mediated amplification of RNA technology (SMART), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), etc. [61]. Under the action of the specific enzymes, the target fragments could be amplified at constant temperature (30e70 C). The procedure only requires a simple and inexpensive device such as a water bath or heating block instead of precise and expensive temperature control instruments used in PCR-based methods such as QuantStudio 3 Real Time PCR System. LAMP is the most popular method among the isothermal amplification methods. Two sets of primers spanning 6 distinct sequences of the target DNA are used to achieve high specificity. LAMP is commonly applied in detection of pathogenic bacteria, viruses and parasites as well as genetically modified organisms (GMO) [46,62]. But there are few explorations in peanut allergen

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detection based on LAMP. In 2018, a LAMP assay was developed for the detection of allergic peanut targeting the Ara h1 gene sequence and the internal transcribed sequence 1 (ITS1) of nuclear ribosomal DNA sequence regions [44]. The amplification reaction was reacted at 60e65 C for 60 min in a heating block and gel electrophoresis was used to detect the LAMP product. The results demonstrated that the sensitivity of LAMP for detecting peanut was higher than the traditional PCR method. Moreover, it showed higher sensitivity by targeting ITS1 than targeting Ara h1 gene (Fig. 7). By integrating LAMP with a microfluidic chip, Dan et al. developed a method to detect the allergen genes of peanut, sesame and soybean [63]. The aim of microfluidic chip technology is to integrate each functional module into a single device to achieve “lab on a chip” (Fig. 8A). In this study, the only equipment required was a simple centrifuge and a thermostatically-controlled water bath. By using a kind of pH indicator, NeuRed dye, a colorimetric method was also developed to indicate a result by a color change which was suitable for the naked eye. During the LAMP reaction, a lot of hydrogen ions were produced and the solution gradually became acidic so that the color would change, otherwise it maintained its original color (Fig. 8B, C). However, as the reaction system contained buffer solution, the pH change was not sufficiently visible. This might explain why the sensitivity of the established LAMP system was not as high as the PCR system. Even so, the method was very convenient and could distinguish the results by naked eye. It is a promising method to detect food allergens on the market. Although there are many advantages of LAMP assay, the design of the primer sets could be complicated due to the coverage of 6 regions of the target sequence. As far as we know, no other isothermal amplification methods of peanut allergy detection are available at present. With the application of isothermal amplification technology in other fields, such as GMO detection based on RPA [45], there is huge potential for isothermal amplification methods to be applied in peanut allergen detection since the trend of detection methods is rapid, convenient and instrument-free.

Fig. 7. The sensitivity of PCR (A, C) and LAMP (B, D) for the detection of peanut targeting ITS1 and Ara h1 gene respectively. Lanes M and N represent 100 bp of DNA ladder and the negative control, respectively. Lanes 1e9 represent the addition of different amounts of peanut DNA: 1, 100 ng; 2, 10 ng; 3, 1 ng; 4, 100 pg; 5, 10 pg; 6, 1 pg; 7, 100 fg; 8, 10 fg; and 9, 1 fg. Black arrow represents the amplified specific PCR product in the agarose gel. Reprinted from ref. 44 Copyright 2018 Elsevier Ltd.

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Fig. 8. Structure of the microfluidic chip (A) and results of the colorimetric test for the sensitivity of the LAMP-microfluidic chip detection method (B, C). Reprinted from ref. 63 Open Access.

3.2. DNA hybridization-based assay Apart from nucleic acid amplification-based method, DNA hybridization-based method, represented by the DNA biosensor, is also a kind of commonly used method for allergen detection [64]. A DNA biosensor is an integrated receptor-transducer device that could recognize the target DNA fragment and convert the recognition event into a measurable chemical or physical signal [65]. It does not need time-consuming amplification procedure and could probably provide a simple, instant, reproducible and cheap portable detection. Compared with nucleic amplification-based method, a DNA biosensor might be a bit more specific since no

non-specific amplification or primer dimer would occur and aerosol pollution would avoid as a second-operation was not required. According to the transduction-based classification, biosensors are divided into electrochemical, optical and other biosensors. Here, several DNA biosensors applied in peanut allergen detection will be introduced. 3.2.1. Electrochemical-based method Due to its low cost and ease of miniaturization, electrochemical DNA biosensors have been widely used for specific gene detection and have great promise for particular applications in rapid food allergen detection. Recent researches were focused on the

Fig. 9. Schematic illustration of the electrochemical DNA sensor by using a stem-loop probe for peanut allergen detection. Reprinted from ref. 66 Copyright 2012 American Chemical Society.

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electrode material and modification to improve the selectivity and sensitivity. Sun et al. developed a novel electrochemical DNA sensor by using a stem-loop probe for peanut allergen Ara h 1 detection [66]. A stem-loop structured DNA probe is superior to linear probe in several aspects for the detection of DNA for the good mismatched

287

discrimination ability. In this study, researchers modified the probe with a thiol at its 50 end and a biotin at its 3'end so that the probe could be self-assembled on the surface of gold electrodes through facile-thiol affinity. 6-Mercaptohexanol (MCH) was used to cover the remnant bare region. When the target DNA was hybridized with

Fig. 10. Schematic diagram of improving the stem-loop DNA biosensor based on grapheneegold nanoparticles (A) and chitosan-mutiwalled carbon nanotube nanocomposite/ spongy gold film (B). Reprinted from ref. 67 Copyright 2014 Elsevier B.V. and from ref. 68 Copyright 2014 Elsevier Ltd.

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the probe, the conformation of the probe would change and the biotin would be detached from the electrode changing the electrontransfer efficiency, which could be detected by electrochemical impedance spectroscopy (EIS) (Fig. 9). The method showed high sensitivity since the detection limit reached 0.35 fM with a linear response ranging from 10
15 to 10
10 M. In 2015, the same group proposed two methods based on different electrode modification materials to improve the performance of the DNA biosensor [67,68]. One was to electrodeposit a multilayer grapheneegold nanocomposite onto a glassy carbon electrode which held good dispersion ability and could amplify the electrochemical signal for its high electron-transfer efficiency (Fig. 10A). Graphene has unique two dimensional structure so it exhibits a large specific surface area, high electrical conductivity which enable it become a promising nanomaterial used in detection field. Gold nanoparticles are also ideal nanomaterial with good conductivity, biocompatibility and electrocatalytic activity. After the electrode modification, the biosensor showed high sensitivity, a fast response time, a wide calibration range and good long-term stability. The other one was to use chitosan-mutiwalled carbon nanotube nanocomposite and spongy gold film to modify the probe immobilization platform, which could increase the effective area (Fig. 10B). Besides, enzyme amplification technology was used to improve the sensitivity. When target DNA was captured by the probe, the change of conformation would trigger a specific interaction between biotin and streptavidin horseradish peroxidase (HRP-SA). Then the target DNA was confirmed by electrochemical detection of the enzymatic product in the presence of substrate. pez et al. In addition to the biosensors described above, Lo developed an electrochemical genosensor that targeted a specific sequence encoding part of Ara h 2 [69]. Screen-printed gold

electrodes were used as the probe immobilization platform. And the biosensor was based on a sandwich format that allowed shortening the capture probe and improving the selectivity (Fig. 11). By using the Design of Experiments (DoE) approach, the genosensor was optimized with a linear range from 5  10
11 to 5  10
8 M and a detection limit of 10 pM. Although the researches above declared that the biosensors had good reproducibility and stability, they still had nearly 20% decline comparing with their initial response [66e68]. The sensors needed to be stored in a refrigerator at 4 C. Besides, the results of electrochemical biosensors are greatly affected by the reaction condition, e.g. pH, temperature, bio-recognition element, and etc. [65]. And the fabrication of the sensor with a good performance is uneasy to achieve in the laboratory, let alone in mass production. Thus, their application in the clinical diagnosis of peanut allergens is still in the research stage. 3.2.2. Optical biosensor-based method Optical biosensors are based on the changes of optical signals such as fluorescence, color, and refractive-index when the target is recognized [70]. The results could be observed by naked eyes. In 2011, Wang et al. developed a silicon-based optical thin-film biosensor chip to detect eight food allergens including peanut [71]. Aldehyde-labeled probes were arrayed and covalently linked to the surface of the biosensor. The target DNA was modified by biotin and when it was captured, the interference pattern of light reflected from the surface changed from gold to purple (Fig. 12). But the sensitivity of the system was only tested on cashew DNA. Another optical biosensor applied for peanut allergen detection was proposed by Maquieira and his co-workers [72]. They developed a DNA microarray method based on a digital versatile disk

Fig. 11. Schematic of genosensor for peanut allergen Ara h 2 detection. Reprinted from ref. 69 Copyright 2014 Elsevier B.V.

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Fig. 12. Food allergen detection on a chip with capture probes spotted by a computer-controlled dispenser. Each spot comprised 50 nL of 10 mmol/L probe solution and was printed in the order shown in (A). (B) Detection of the food allergenspecific genes on thin-film biosensor chips: 1, blank control (ddH2O); 2, cashew; 3, peanut; 4, wheat; 5, soybean; 6, chicken; 7, fish; 8, shrimp; 9, beef. Reprinted from ref. 71 Copyright 2011 American Chemical Society.

(DVD) for simultaneous detection of trace hazelnut, peanut and soybean in food products (Fig. 13). Utilizing the change of the signal intensity of the laser that reached the DVD drive, it achieved the visual detection. The assay was proposed as a specific, sensitive and suitable analytical tool to detect the three target allergens. But the target templates of these two methods above were come from PCR amplification products. The target DNA was modified during the amplification procedure. It did not save time and the drawbacks of PCR still existed. But with the advancement of PCR methods, these limitations could be overcome [12]. It is worth noticing that this kind of methods lays the foundation for a novel high-throughput method for simultaneous detection of multiple allergens in foods. Compared with multiple-PCR method, it has higher sensitivity and fast response time.

The biggest advantage of DNA strand hybridization-based methods is that they enable higher efficiency and sample throughput. The modification of nanomaterials has led to improved sensitivity, selectivity, robustness and accuracy of detection methods. However, for ultra-trace allergens, he DNA strand hybridization-based methods are inferior in sensitivity to the nucleic acid amplification-based methods for the latter could perform a million-fold amplification of trace DNA fragments [62]. Other challenges are how to enhance the signal-to-noise ratio, signal amplification and enhancement of the transduction efficiency [73]. Thus, DNA strand hybridization-based methods still need to improve by optimizing operation platform and developing novel modification methods of target DNA, aiming to develop a rapid, portable, cost-effective detection.

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Fig. 13. (A) Scheme of DVD arrays. (B) Scheme of the assay format developed on DVD surface. (C) Optical density images from different assays. Panels AeF correspond to 0, 0.1, 1, 10, 100, and 1000 mg/g of allergen mixture (hazelnut, peanut, and soybean), respectively. Reprinted from ref. 72 Copyright 2012 American Chemical Society.

4. Conclusions and prospects Since peanut allergy has grown more and more concern over the world and the mainstream way to prevent allergic reaction is to avoid exposure to peanut allergens, it is greatly necessary and urgent to develop a rapid, convenient and accurate method to detect peanut allergens in processed food. As the large number of allergenic foods that require mandatory ingredient labeling or have potential cross-contamination, DNA-based methods could be the better choice than protein-based methods. Nucleic acid amplification methods would still play an indispensable role in the future due to the trace amount of allergens in processed food. PCR methods have been successfully applied for food allergen detection for their high specificity, sensitivity and rapidity, but they are mainly limited by the presence of inhibitors [74]. As DNA isolation methods improves, it is possible to remove the several inhibitor compounds of the amplification reaction. The digital PCR method would get more attention for its absolute quantification ability and simple device [75]. Besides, kind of isothermal amplification such as LAMP, RPA would offer a significant breakthrough for on-site detection of food allergy. On the other hand, DNA hybridizationbased methods would enable an easy-to-use detection system for a number of food allergens since the reaction process is rapid and analyze platform is easy to be miniaturized. Another important advantage of DNA-based methods is that the diagnosis system of peanut allergen could be applied to other food allergens. The principle of establishing detection system is similar.

For different food allergens, we screen for the appropriate DNA fragments through the allergen genome. The technological advances are continuously offering novel approaches for analysis and detect allergens in processed food. We have reasons to believe that the detection of food allergens will become easier and cheaper. By developing and optimizing these advanced detection methods, not only food-manufacturing companies but also ordinary families in need can achieve rapid allergen detection before selling or consuming commercial food products. Acknowledgement This work was supported by the National Natural Science Foundation of China (31571918) and Key Research and Development Program of Yunnan Province (2018BC005). References [1] S.H. Sicherer, H.A. Sampson, Food allergy: a review and update on epidemiology, pathogenesis, diagnosis, prevention, and management, J. Allergy Clin. Immunol. 141 (2018) 41e58. https://doi.org/10.1016/j.jaci.2017.11.003. [2] H.A. Sampson, Food allergy: past, present and future, Allergol. Int. 65 (2016) 363e369. https://doi.org/10.1016/j.alit.2016.08.006. [3] S.K. Sathe, C. Liu, V.D. Zaffran, Food allergy, Annu. Rev. Food Sci. Technol 7 (2016) 191e220. https://doi.org/10.1146/annurev-food-041715-033308. [4] Report of the FAO Technical Consultation on Food Allergies, Food, Agricultural Organization of the United Nations, Rome, Italy, 1995. [5] Z. Husain, R.A. Schwartz, Peanut allergy: an increasingly common lifethreatening disorder, J. Am. Acad. Dermatol. 66 (2012) 136e143. https://doi. org/10.1016/j.jaad.2011.02.031.

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