Development of detection method for edible silkworm (Bombyx mori) using real-time PCR

Food Control 94 (2018) 295–299

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Food Control journal homepage: www.elsevier.com/locate/foodcont

Development of detection method for edible silkworm (Bombyx mori) using real-time PCR

T

Mi-Ju Kim, Seul-Ki Jung, Sung-Yeon Kim, Hae-Yeong Kim∗ Institute of Life Sciences & Resources and Department of Food Science and Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Silkworm Edible insect Real-time PCR Cytochrome oxidase subunit I Processed food

The consumption of silkworms as edible insects has recently increased because of their good nutrition content. Many insect-based products are processed into a powder because of their unappetizing appearance. In this aspect, powdered silkworm has the possibility to be mixed with other insects. Thus, the development of a detection method for silkworm is necessary to provide accurate label information to consumers. In this study, we developed a real-time PCR assay using a TaqMan probe for detection of silkworm. The newly designed silkwormspecific primer pair and probe target the mitochondrial cytochrome oxidase subunit I (COI) gene. They were tested for their specificity using 15 insect species, and were confirmed to amplify only silkworm species. The limit of detection of this method was 0.001 ng of silkworm DNA, and as little as 1% of silkworm in two or more insect mixtures was detected. This developed method was applied to 30 processed foods including edible insects and was validated with four different real-time instruments. Therefore, the silkworm-specific real-time PCR assay may provide a specific and sensitive method for detection of silkworm in raw and processed foods.

1. Introduction The world population is expected to reach 9 billion by 2050, and many researchers, consumers, and food technologists are focusing on novel and sustainable foods for the future. According to a report by the Food and Agricultural Organization (FAO) of the United Nations, insects are promising future nutrition sources for people in regard to their environmental and economic aspects (Tomotake, Katagiri, & Yamato, 2010; Van Huis et al., 2013). Insects for human consumption not only provide high levels of protein, vitamins, minerals, and fat, but also have a higher feed-conversion efficiency than meat served as an animal protein source (Belluco et al., 2013; Van Huis et al., 2013). Many species of insects have been traditionally eaten in Africa, Asia, and Latin America, and some Western countries have also accepted edible insects (Srinroch, Srisomsap, Chokchaichamnankit, Punyarit, & Phiriyangkul, 2015; Ulrich et al., 2017). For example, in the Netherlands, insects from the Tenebrionidae family such as mealworm and super mealworm has been raised as feed, and in the USA, the house cricket (Acheta domesticus) has been used in some processed foods such as energy bars and dried foods (Rumpold & Schlüter, 2013; Van Huis et al., 2013). The Agence féderale pour la sécurité de la chaîne alimentaire (AFSCA) of Belgium announced that the edible insect list contains 10 species of insects, and the Ministry of Food and Drug Safety (MFDS) in Korea

∗

authorized seven edible insects including silkworm as acceptable food ingredients (Ghosh, Lee, Jung, & Meyer-Rochow, 2017). Among the various species of edible insects, silkworms have been used as medical raw materials in traditional Korean medicine, leading to a greater acceptance in Korean food (Ghosh, Lee, Jung, & MeyerRochow. 2017; Ryu, Kim, Ahn, Kim, & Lee. 2003). In addition, silkworms have already been domesticated because of their commercially valuable pupae as well as their silk (Tomotake et al., 2010). Because the silkworm protein is known to contain many essential amino acids such as valine and methionine, the edible silkworm has been introduced as a good nutrition source for humans in a variety of preparation methods. However, despite utilization of edible insects as a nutrition source, their unappetizing appearance makes some consumers hesitant about buying them. To resolve this problem, insects for food consumption can be ground into powder and mixed with other food ingredients (Van Huis et al., 2013). However, this can increase the possibility of mixing insect species into a food without declaring it on the products’ label (Ulrich et al., 2017). Thus, it is important to be able to accurately detect edible insect components in processed products, especially because silkworm could be a food allergy (Srinroch et al., 2015). Because insectbased foods may be processed with boiling, roasting, and/or frying to ensure food safety in regard to microbial survival and growth (Klunder, Wlkers-Rooijackers, Korpela, & Nout, 2012), detection methodologies

Corresponding author. E-mail address: [email protected] (H.-Y. Kim).

https://doi.org/10.1016/j.foodcont.2018.07.021 Received 15 February 2018; Received in revised form 12 June 2018; Accepted 14 July 2018 Available online 19 July 2018 0956-7135/ © 2018 Elsevier Ltd. All rights reserved.

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Fig. 1. Sequence alignment of the cytochrome C oxidase subunit I (COI) gene against 26 insect species and selection of silkworm specific primers and probes (1–26: Bombyx mori, Galleria mellonella, Plodia interpunctella, Biston betularia, Tineloa bisselliella, Tenebrio molitor, Allomyrina dichotoma, Protaetia brevitarsis, Zophobas atratus, Promethis valgipes, Heterotarsus carinula, Gonocephalum pubens, Dorcus titanus castanicolor, Prosopocoilus inclinatus, Dorcus rubrofemoratus, Oxya chinensis, Locusta migratoria, Atractomorpha lata, Tetrix japonica, Shirakiacris shirakii, Acrida cinerea, Loxoblemmus campestris, Velarifictorus micado, Teleogryllus emma, Gryllus bimaculatus, and Apis mellifera).

species (Bombay locust and honey bee pupa) to evaluate the effect of different insects in mixed samples on this assay. Insects for reference insect mixtures were freeze-dried and ground. Each insect mixture was measured to a final weight 100 mg for DNA isolation. A total of 30 processed products including hot air- and freeze-dried, fried, and canned insect foods were purchased from farms, local markets, and online shops in Korea, Thailand, and the USA.

must be sensitive and specific, and account for these potential treatments. Usually, insect analysis methods are used for genetic classification using DNA barcodes based on the cytochrome C oxidase subunit I (COI) gene, and for morphological classification using insect shape, texture, and color (Wena & Guyer, 2012). The methods for detecting and discriminating between insects have been used mainly to compare phylogenetic and biological information (Choi et al., 2015; Park et al., 2013). Recently, a method for the identification of edible insects using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was developed targeting insect protein (Ulrich et al., 2017), and a real-time PCR assay for detecting mealworm from food and feed has also been proposed (Debode et al., 2017). Both proteinand DNA-based approaches have been used mainly for the detection of food components. However, the thermal stability of the DNA molecule lends real-time PCR to be more practical than protein-based detection methods. Nevertheless, to our knowledge, no method has been proposed to distinguish silkworms specifically from other insects and to detect them in processed foods using silkworm-specific real-time PCR system. Therefore, in this study, silkworm-specific primers and probes were designed targeting the silkworm mitochondrial COI gene, and a real-time PCR assay using a TaqMan probe was developed for authentication of the silkworm species. The applicability of this detection method was verified for silkworm species in a variety of raw and processed products.

2.2. DNA extraction Each sample for DNA extraction had the intestine removed and was then ground in liquid nitrogen. Then, DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions with a small modification. Briefly, 20 mg of raw and processed food samples were lysed, and all buffers specified in the manufacturer's protocol were used at double the quantity to obtain DNA with good purity. DNA concentration and purity were measured using Maestro Nano Spectrophotometer (Maestrogen, Las Vegas, NV, USA) and DNA with a 260/280 nm ratio between 1.7 and 2.0 was used as template DNA. 2.3. Primer and probe design The sequences of the COI region for silkworm and other insects were acquired from the GenBank database (Tables S–1) and then compared using the Clustal Omega alignment system (Fig. 1). Silkworm-specific primers and the probe were designed by Primer Designer, version 3.0 (Scientific and Educational Software, Durtham, NC, USA), and synthesized by Bionics (Seoul, Korea) and Bioneer (Daejeon, Korea), respectively. Fig. 1 and Table 1 provide information on the primers and probes used in this study.

2. Materials and methods 2.1. Samples A total of 15 insect species including silkworm, mealworm, twospotted cricket, rhinoceros beetle larvae, white spotted flower chafer beetle larvae, rice grasshopper, migratory locust, super-mealworm, oriental garden cricket, honey bee pupa, Dae-wang darkling beetle, Allak cricket, Manchurian spotted chafer beetle, stag beetle, and pygmy locust were used for the experiment. All insect samples were obtained from the Rural Development Administration (RDA) and the National Institute of Biological Resources (NIBR) in Korea. Each insect was washed with distilled water and immediately stored in a freezer tube at −20 °C until analysis. Two independent sets of reference insect mixtures containing 50, 10, and 1 (w/w) of silkworm were prepared using three insect species (mealworm, two-spotted cricket, and rice grasshopper) and two insect

2.4. Conventional PCR and real-time PCR conditions Conventional PCR was performed using a Thermocycler PC808 (ASTEC, Fukuoka, Japan), and a total amount of conventional PCR mixture containing silkworm-specific primer was 25 μl. The conventional PCR mixture consisted of 1 μl (10 ng/μl) of sample DNA, 2.5 μl of 10x buffer (Takara, Japan), 2 μl of dNTPs (each dNTP at 2.5 mM, Takara), 0.1 μl of ex Taq DNA polymerase (5 units, Takara), and 1 μl of each primer (0.4 μM). Conventional PCR amplification was carried out as follows: pre-denaturation for 1 cycle at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, with a final 296

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Table 1 The primers and probes used in this study. Primer name

Sequence (5’→ 3′)

PCR product size (bp)

Target genes

Reference

BMCOI-F BMCOI-R BMCOI probe 18S-INS-2F 18S-INS-R 18S-INS probe

TCC TAC CCC CCT CCC TTA TA GCA AGA TCT ACG GAT CTT CC FAM-GTG CAG GAA CAG GAT GAA CA-TAMRA GCG ACG GAT CTT TCA AAT GTC CCC CGT TAC CCG TTA CAA CC FAM-CTT ATC AAC TGT CGA TGG TAG GTT CTG CGC-TAMRA

127

COI

This study

81

18S rRNA

Debode et al. (2017)

listed in Table 1. To evaluate the specificity of the newly designed silkworm-specific primer, conventional PCR was performed using 15 insect species (silkworm, mealworm, two-spotted cricket, rhinoceros beetle larvae, white spotted flower chafer beetle larvae, rice grasshopper, migratory locust, super-mealworm, oriental garden cricket, honey bee pupa, Daewang darkling beetle, Al-lak cricket, Manchurian spotted chafer beetle, stag beetle, and pygmy locust), as shown in Fig. S-1. The silkwormspecific primer specifically amplified silkworm DNA without any cross reactivity against other insects (Fig. S-1). The real-time PCR assay for the detection of silkworm was developed using a TaqMan probe designed with silkworm sequences between forward and reverse primers. The specificity of this assay was also tested with the 15 insect species, and a universal primer targeting an 18S rRNA gene was additionally used as an endogenous control to verify the presence of DNA extracted from the 15 insects. As shown in Table 2 and Fig. S-2, only target species were amplified by the silkworm-specific primer/probe, whereas amplifications of all insect species occurred with the universal primer/probe. These results show that the silkworm-specific real-time system was specific to the target species.

extension at 72 °C for 5 min. All PCR products were electrophoresed on a 2% agarose gel in 0.5x Tris-acetate-EDTA buffer, and then stained with ethidium bromide. Real-time PCR was performed using ABI 7500 (Applied Biosystems, Foster City, CA, USA), and the total amount of real-time PCR mixture containing silkworm-specific primer/probe was 25 μl. The real-time PCR mixture consisted of 1 μl of sample DNA (10 ng/μl), 12.5 μl of 2x TaqMan® Universal Master Mix (Applied Biosystems), 1 μl of probe (0.2 μM), and 1 μl of each primer (0.4 μM). Real-time PCR amplification was carried out as follows: 1 cycle of 94 °C for 10 min, 40 cycles of 15 s at 95 °C, and 1 min at 56 °C. Each PCR reaction was performed in triplicate, and a negative control was used with a non-template sample in all reactions. 2.5. Specificity and sensitivity tests The specificity of the silkworm-specific primer in both conventional and real-time PCR assays was tested using DNA extracted from 15 different insects. To confirm DNA extracted from all insects, a universal primer based on the 18S rRNA gene was also performed as the endogenous control. The sensitivity of the silkworm-specific real-time PCR was evaluated using diluted DNA (10, 1, 0.1, 0.01, 0.001, and 0.0005 ng) isolated from raw silkworm. To determine the absolute limit of detection (LOD) for silkworm detection, each sample was assayed with 10 replicates. Furthermore, two independent sets of reference insect mixtures were also used for the sensitivity test of this assay.

3.2. Linearity and sensitivity results of real-time PCR To evaluate the efficiency and linearity of the silkworm-specific real-time PCR assay, a standard curve was constructed using 10-fold serial dilutions of silkworm DNA ranging from 10 ng to 1 pg. The slope and correlation coefficient (R2) of the standard curve were −3.59 and 0.996, respectively, and PCR efficiency (%), which was calculated using the equation E = [10(−1/slope) - 1] was 89.97 (Table 3). These values indicate good linearity and high efficiency for silkworm detection. The

2.6. Validation of real-time PCR The developed real-time PCR method was validated using different real-time PCR instruments - ABI 7500, ABI 7500 fast, StepOnePlus, and ViiA 7 (Applied Biosystems). The four different real-time PCRs were evaluated constructing a standard curve of silkworm-specific primer/ probe, and then slope and R2 obtained by each instrument were evaluated according to the European Network of GMO Laboratories guidelines (ENGL, 2015). A total of 30 processed products were also applied to verify the reliability of this assay and assess the variation of instruments.

Table 2 Specificity results using the real-time PCR system.

3. Results and discussion 3.1. Specificity results The COI gene sequences of 26 different insects including four Lepidoptera (Galleria mellonella, Plodia interpunctella, Biston betularia, and Tineloa bisselliella) were aligned to select a silkworm-specific region (Fig. 1 and Tables S–1). The silkworm-specific primers and probes were designed having a PCR product size of 127 bp from positions 302–428 (Accession No. NC_ 002355.1) on the COI region. A short amplicon (< 150 bp) can improve the possibility of identifying silkworm in processed food products because DNA is broken down into small fragments under high temperature and pressure (Ali et al., 2015; Arslan, Ilhak, & Calicioglu. 2006; Haunshi, Basumatary, Girish, Doley, Bardoloi, & Kumar. 2009; Kim & Kim. 2017). The primer and probe information used in this study is

Common name

Scientific name

Insectsspecific PCR

Silkwormspecific PCR

Silkworm Two-spotted cricket Mealworm Rhinoceros beetle larvae White spotted flower chafer beetle larvae Rice grasshopper Migratory locust Super-mealworm Oriental garden cricket Honeybee pupa Dae-wang darkling beetle Al-lak cricket Manchurian spotted chafer beetle Stag beetle

Bombyx mori Gryllus bimaculatus Tenebrio molitor Allomyrina dichotomus

+ + + +

+ – – –

Protaetia brevitarsis

+

–

Oxya chinensis Locusta migratoria Zophobas atratus Teleogryllus emma Apis mellifera ligustica Prometheus valgipes

+ + + + + +

– – – – – –

Loxoblemmus campestris Protaetia mandschuriensis Lucanus maculifemoratus dybowskyi Tetrix japonica

+ +

– –

+

–

+

–

Pygmy locust

297

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0.001 ng of silkworm DNA was subjected to this method, positive signals were observed in all 10 replicates (Fig. S-3). However, when 0.0005 ng of silkworm DNA was applied using this method, only 7 out of 10 positive signals were detected (Fig. S-3). Thus, the absolute LOD of the developed silkworm-specific real-time system was found to be 0.001 ng. Reference insect mixtures were additionally tested to evaluate the effect of different insects in silkworm-specific real-time PCR and determine the sensitivity of this assay. As shown in Table 4, the developed method in this study successfully amplified silkworm even though it was mixed with two or more insects. In two independent sets of reference mixtures, as little as 1% silkworm was detected (Table 4), and the sensitivity of this assay was sufficient for the authentication of silkworm in edible insect foods.

Table 3 Inter-laboratory validation results of the real-time PCR system using standard curves. Real-time PCR instruments

Efficiency R2 Slope

ABI 7500

ABI 7500 fast

StepOnePlus

Viia 7

89.97 0.996 −3.59

89.88 0.999 −3.59

89.81 0.998 −3.59

99.12 0.997 −3.34

All PCR reactions for construction of a standard curve were performed in triplicate. Table 4 Results of silkworm-specific real-time PCR in reference insect mixtures. Reference insect mixtures

Silkworm in 3 insect species (Mealworm, two-spotted cricket, and rice grasshopper) Silkworm in 2 insect species (Bombay locust and honey bee pupa) a

Silkworm content (%)

Ct values obtained silkworm-specific realtime PCR

50 10 1 50 10 1

22.45 24.29 27.14 22.79 24.19 26.61

± ± ± ± ± ±

3.3. Analysis of processed food containing edible insects A total of 30 processed foods containing silkworm or other edible insects (mealworm, two-spotted cricket, rice grasshopper, and Bombay locust) were selected to confirm the applicability of the developed realtime PCR assay. Because silkworm DNA can be degraded at high temperature and pressure, it is important to apply this method to a variety of processed samples. Thus, our selection of foods included hot air- and freeze-dried, fried, and canned edible insect foods. Table 5 shows the results of real-time PCR for silkworm identification in 30 processed foods. 25 samples that declared silkworm on the product label were detected by the silkworm-specific real-time PCR assay, and no amplification was observed in five samples that did not contain silkworm (Table 5). This result shows that it is sufficient to detect silkworm species in highly processed products as well as raw silkworm samples.

0.06a 0.23 0.29 0.07 0.07 0.13

Mean Ct value ± SD obtained from triplicate reactions.

silkworm-specific primers and probe developed in this study were acceptable according to the ENGL criteria, where the slope should be in the range of −3.1 ≤ slope ≥ −3.6 and R2 value should be ≥ 0.98. The sensitivity of the developed real-time PCR assay was measured using dilutions of silkworm DNA (10, 1, 0.1, 0.01, 0.001, and 0.0005 ng), and each sample was assayed with 10 replicates. When

Table 5 Results of silkworm identification in processed foods using four different real-time PCR instruments. No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 a b

Processing/product type

Drying/globule Drying/globule Drying/globule Drying/globule Freeze-drying/globule Drying/globule Drying/powder Freeze-drying/powder Freeze-drying/powder Drying/powder Drying/powder Drying/powder Drying/powder Drying/powder Drying/chrysalises Drying/chrysalises Drying/chrysalises Drying/chrysalises Drying/chrysalises Drying/chrysalises Drying/chrysalises Drying/chrysalises Can/chrysalises Drying/chrysalises Frying/chrysalises Drying/powder Drying/powder Drying Drying Drying

Silkworm content

70% 49% 38% 90% 100% 35% 100% 99.5% 100% 100% 100% 100% -a 100% 100% 100% 100% 100% 100% 100% 100% -a 70% -a 100% 0% 0% 0% 0% 0%

Silkworm origin

Korea China Korea Korea Korea Korea Korea Korea Korea China Korea Korea USA Thailand Korea China Korea Korea Korea Korea Thailand USA China Thailand Thailand NA NA NA NA NAb

Other ingredients

Mulberry Mulberry Mulberry Mulberry

leaves leaves leaves leaves

Mulberry leaves

Chocolate Mealworm Mealworm and two-spotted cricket Two spotted cricket Rice grasshopper Bombay locust

Silkworm species were labeled on products but accurate contents were not provided. Not applied. 298

Real-time PCR instruments ABI 7500

ABI 7500 fast

StepOnePlus

ViiA 7

+ + + + + + + + + + + + + + + + + + + + + + + + + – – – – –

+ + + + + + + + + + + + + + + + + + + + + + + + + – – – – –

+ + + + + + + + + + + + + + + + + + + + + + + + + – – – – –

+ + + + + + + + + + + + + + + + + + + + + + + + + – – – – –

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3.4. Validation results of real-time PCR

doi.org/10.1016/j.foodcont.2018.07.021.

To estimate the reliability of the developed real-time method, a validation study was performed with four different real-time PCR instruments (ABI 7500, ABI 7500 fast, StepOnePlus, and ViiA 7). Validation of the assay with various instruments can provide assurance that this assay will be effective in many different laboratories (Debode et al., 2017). First, a standard curve was constructed with each of the four instruments using 10-fold diluted silkworm DNA (range from 10 ng to 1 pg), and these results are summarized in Table 3. Considering the ENGL guidelines described above, slope and R2 values of the standard curve were satisfied in all systems (Table 3). For an additional validation experiment, the four real-time PCR instruments were used to assay the 30 processed foods containing edible insects; all four instruments detected silkworm in only 25 processed foods that declared it on the product label (Table 5). Thus, the silkworm-specific real-time PCR assay developed in this study is a reliable method for the identification of silkworm and is suitable for silkworm food monitoring. Recently, DNA- and protein-based methods for the detection of edible insects have been employed (Debode et al., 2017; Ulrich et al., 2017). However, there was not a detection and monitoring method for edible insects in processed products. Specifically, to our knowledge, this is the first sensitive, specific, and reliable real-time PCR method for silkworm detection in various processed products. Therefore, this study increases the applicability of method development for other edible insect detection using real-time PCR.

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4. Conclusion With the increasing interest in edible insects in the food industry, it is critical to authenticate insect species mixed in a food matrix. In this study, a real-time PCR assay based on the TaqMan probe was developed to identify edible silkworm species in raw and processed food samples. Furthermore, for the validation study, different real-time instruments were applied and they all successfully detected silkworm species in various types of processed products. Therefore, this method can be utilized for detecting the presence of silkworm species in the field of insect-based food control. Acknowledgments This research was supported by grant 17162MFDS065 from the Ministry of Food and Drug Safety in Korea. Appendix A. Supplementary data Supplementary data related to this article can be found at https://

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