Development of loop-mediated isothermal amplification (LAMP) assay for rapid and direct screening of yellowfin tuna (Thunnus albacares) in commercial fish products.

Tuna is one of the most widely consumed fish on the European market, being available in various consumable options. Among them, Thunnus albacares, also called yellowfin tuna, is a delicacy and is consumed by millions of people around the world. Due to its comparatively high cost and demand, it is more vulnerable to fraud, where low-cost tuna or other fish varieties might be replaced for economic gain. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for targeting the mitochondrial cytochrome b gene for fast and direct detection of Thunnus albacares, which is a valuable tuna species. The analytical specificity was confirmed using 18 target samples (Thunnus albacares) and 18 samples of non-target fish species. The analytical sensitivity of the LAMP assay was 540 fg DNA per reaction. In addition, a simple and direct swab method without time-consuming nucleic acid extraction procedures and the necessity for cost-intensive laboratory equipment was performed that allowed LAMP detection of Thunnus albacares samples within 13 minutes. Due to its high specificity and sensitivity, the LAMP assay can be used as a rapid and on-site screening method for identifying Thunnus albacares, potentially providing a valuable monitoring tool for food authenticity control by the authorities.

Introduction

Tuna belongs to the Thunnus tribe (scombridae) family, and it is one of the most popular delicacies among marine fish globally. Tuna is a very large species which is widely distributed in oceanic environments. It is consumed in various forms, either fresh or as canned products. The Council Regulation (EEC) No. 1536/92 defines relevant members of the genus Thunnus on the European market, which include yellowfin tuna (Thunnus (neothunnus) albacares) and skipjack tuna (Euthynnus (Katsuwonus) pelamis). These two species dominate the market share with more than 80%, with yellowfin tuna belonging to the higher price category [1].

Tuna is considered as a healthy source of animal proteins, “omega-3” fatty acids and bioactive nitrogen compounds, which make it more popular among many fish species. Due to its bulky size, it is not possible to sell tuna as one piece so that it is usually traded in portions or types of fillet. Alternatively, it is offered as a floss product after being dried, flavoured, roasted and rolled. Tuna is mainly used for canning and making sashimi and sushi products. The characteristic texture, taste and flavour of canned tuna products, available either in oil or brine, makes it a delicacy for consumers. Among the designated tuna species, yellowfin tuna (Thunnus albacares) is one of the most widely used species in canned products.

Prices for different tuna species vary according to species and popularity. The prices are regulated by species of tuna, trade performance and ultimate use. The market price of raw tuna, mostly used in sashimi and sushi, is quite high. Sashimi preparation requires high quality fish and usually is the most demanding and expensive option on the market [2]. Compared to fresh and raw tuna, quality requirements are not very stringent for canned, cooked, frozen and smoked tuna products. Prices for skipjack tuna are much lower than for bigeye, bluefin and yellowfin tuna due to market-specific influences and consumption patterns in European countries [1].

There are studies which indicate mislabelling of various tuna products, particularly skipjack and yellowfin tuna [2, 3]. Due to price variations and taxes imposed by the European Union on imported products, attention has been given to methods which can identify canned fish species in accordance with EU regulations. Several methods based on protein and DNA detection are available to differentiate between fish species. For example, raw tuna and other fish products can be identified using the isoelectric focusing (IEF) method, which is based on the availability of water-soluble proteins in the fish species [4]. Proteomics-based approaches like peptide mass fingerprint (MALDI-TOF) and peptide fragment fingerprinting (MALDI-LIFT TOF/TOF) separated by 1D SDS-PAGE have also been applied for tuna species identification [5]. These methods are based on the analysis of proteins present in by-products of canned tuna as well as in products obtained from filleting the skeletal red muscle of fresh fish [5]. Apart from the high labour input involved in performing this procedure, steps like canning, cooking and smoking involve intense heat treatment which can cause irreversible loss of water and affects solubility of proteins, hampering the identification process [6]. Methods based on antibody-antigen interaction can provide suitable alternatives, but until now, only a handful of immunoassays have been developed, and none of them are available for large-scale application for routine analysis. DNA amplification-based methods such as PCR–RFLP [7], PCR-SSCP [8] or real-time PCR [9] as well as sequencing-based methods are also applicable for fish species detection [10]. However, performing PCR requires time-consuming cycling steps, costly and bulky equipment as well as a laboratory environment, thus limiting its utility for on-site or rapid diagnosis.

There are several isothermal amplification methods that can amplify DNA at a constant temperature using a simple water bath or heating block without the need for expensive thermal cyclers and detection systems. The isothermal methods include Strand Displacement Amplification (SDA), Rolling Circle Amplification (RCA), the Ramification Amplification Method (RAM), Isothermal Multiple Displacement Amplification (IMDA), Nucleic Acid Sequence Based Amplification (NASBA), Helicase-Dependent Amplification (HAD), Recombinase Polymerase Amplification (RPA), Polymerase Spiral Reaction (PSR) methods and Loop-Mediated Isothermal Amplification (LAMP). The above have been applied for detecting several infectious and non-infectious diseases of animals and humans. Each method has its advantages and disadvantages [11]. In the past few years, the nucleic acid amplification technique called loop-mediated isothermal amplification (LAMP) has gained increasing popularity. This method can be used for fast identification of different targets, generally offering possibilities for on-site application. LAMP uses four to six specifically designed primers that recognise six to eight regions on the target gene sequence, resulting in high sensitivity and specificity. Additionally, the complete identification process can be performed under isothermal conditions in less than 30 minutes. In the last few years, many studies have been performed using LAMP for food authentication, including eel [12], salmon [13], cod [14], tuna [15] and ostrich meat [16].

The aim of the present study was to develop a new LAMP assay for direct detection of yellowfin tuna (Thunnus albacares) in commercial food products, targeting the mitochondrial cytochrome b gene (cytB).

Material and methods

Sample collection

A total of 36 samples were collected from different supermarkets in Hannover, Germany. Of these, 18 samples were labelled as Thunnus albacares steaks. The remaining 18 non-target samples included six canned samples of Katsuwonus pelamis, two Oncorhynchus keta fillets, one Onchorhynchus nerka fillet, one Salmon salar fillet, five Gadus morhua fillets and three Gadus chalcogrammus fillets. The samples were stored in accordance with the manufacturers’ specifications at -20°C or at room temperature until use. Manufacturer information is available as supplementary data (Table 1s in S1 Data).

DNA extraction

DNA extraction was performed using the DNeasy Blood &Tissue Kit (Qiagen GmbH, Hilden, Germany) in accordance with the manufacturer’s instructions. The concentration and purity of extracted DNA was checked using the spectrophotometer Nanodrop 2000c (Thermo Fisher scientific, VWR International GmbH, Darmstadt, Germany). Extracted DNA was stored at 4°C until use.

Designing of species-specific LAMP primers

The mitochondrial cytochrome b (cytB) gene sequence (accession no. JN086153.1) of Thunnus albacares was retrieved from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and used as a target for species-specific LAMP primer design. In a first step, the template sequence was aligned with several cytB gene sequences of the target species obtained from the National Centre for Biotechnology Information (NCBI) (Bethesda, MD, USA). The alignments were performed using the Basic Local Alignment Search Tool (BLAST) by NCBI and are available as supplementary material (Figure 1s in S1 Data). Homologous regions with 100% coverage of nucleotide bases were selected as potential primer-binding sites. Alignment of cytB genes was also performed with relevant non-target species on the European market like skipjack tuna and other fish species to ensure that cross-reactions would be avoided by sufficient mismatches within the primer binding sites.

Six oligonucleotide primers, including forward and backward outer primers (F3 and B3), forward and backward inner primers (FIP and BIP) as well as forward and backward loop primers (LF and LB), were designed using the LAMP Designer software, ver. 1.10 (PREMIER Biosoft, CA, USA) and synthesised in HPSF purified quality by Eurofins Genomics GmbH (Ebersberg, Germany). Primer sequences are shown in Table 1.

Table 1

cytB gene-targeting LAMP primer sequences.

Designation	Primer Sequence	Length	Position (NCBI acc. no. JN086153.1)
Tuna F3	ATACGCAATTCTTCGGTCC	19 bp	15212–15230
Tuna B3	TTGTTCTCAGCTCAGCCT	18 bp	15488–15505
Tuna LF	GAAGTGTGCAGGAAGGGAA	19 bp	15292–15310
Tuna LB	GCGGAACAGCCCTTCATTA	19 bp	15408–15426
Tuna FIP	TGGTCGGAATGTTAGAGTT-CGCAGCCTCCATCCTTGTACTT	41 bp	15317–15338
			15266–15284
Tuna BIP	TGCAGACGTAGCCATTCTT-ACCAGGCTACTTGGCCGATAA	40 bp	15368–15389
			15427–15444

Analytical specificity and sensitivity of the Thunnus albacares LAMP assay

For determining analytical specificity of the newly established cytB LAMP assay, 18 Thunnus albacares samples and 18 samples of other fish species were comparatively tested with real-time PCR. Analytical sensitivity of LAMP assay was defined by testing 10-fold serial diluted DNA of Thunnus albacares using AE buffer (Qiagen Ltd.) as a dilution medium. Dilutions contained 54 ng/μL to 0.54 pg/μL of DNA. Each dilution was tested in triplicate.

Direct detection by MSwab

For direct detection of Thunnus albacares without elaborate sample processing steps, a swab method was performed using the MSwab kit (Copan S.p.A., Brescia, Italy) [17]. The dry swab was picked from the supplier packet and scrubbed and rolled firmly several times over the surface of two steak samples designated as Thunnus albacares. The swabs were then dipped into the MSwab® buffer tubes with the swab head detached and remaining inside. Tubes were shaken vigorously by hand without any vortexing or incubation. After proper mixing, 5 μL from each buffer tube were directly used as a template for LAMP testing. Besides the regularly run positive and negative control reactions, one kit-extracted sample of Salmon salar (Sal 1) was taken as negative extraction control during LAMP testing of the swab samples.

LAMP assay

LAMP reactions were performed using the portable real-time fluorimeter Genie II® (Optigene Ltd., Horsham, UK). Each reaction mixture with a total volume of 25 μL consisted of 15 μL GspSSD Isothermal Mastermix (ISO-001) (OptiGene Ltd.), 2.5 μL primer mix, 2.5 μL Nuclease-free water and 5 μL template DNA. The primer mix was prepared in a standard version in accordance with the recommendations of OptiGene Ltd. (http://www.optigene.co.uk/support/). In each reaction, the concentration of F3 and B3 primers was 0.2 μM, of FIP and BIP primers 0.8 μM and of LF and LB primers 0.4 μM. The LAMP reaction was performed at 65°C for 3040 minutes followed by melting curve analysis for descending temperatures between 98°C–80°C (ramp rate: 0.05°C/s). In each run, a positive and a negative control reaction were performed using 5 μL of DNA template (0.1 ng/μL) from Thunnus albacares (T4) and 5 μL of nuclease-free water instead of the DNA template, respectively.

PCR assay

A slightly modified real-time PCR assay based on the descriptions of Lee et al. [18] was performed to confirm the analytical specificity of the LAMP assay. Each reaction mixture with a total volume of 30 μL contained 15 μL FastStart Essential DNA Green Master (Roche Diagnostics GmbH, Mannheim, Germany), 1 μL of Alba-F and Alba-B primers (10 μM) each, 10 μL PCR gradient water and 3 μL DNA template. The thermocycling process was started with a preincubation step for 10 min at 95°C, followed by 35 three-step amplification cycles, each including 30-s periods of denaturation at 95°C, annealing at 64°C and extension at 72°C. Subsequently, a final extension step was performed at 72°C for 5 min. Amplification was followed by melting curve generation at 95°C for 10 s, 65°C for 60 s and 97°C for 1 s. All PCR reactions were carried out using the real-time LightCycler96 system (Roche Diagnostics GmbH). Primers were ordered in HPSF purified quality (Eurofins Genomics GmbH).

Gel electrophoresis

In addition to evaluating the melting temperatures, the specificity of the Thunnus albacares LAMP products obtained from 10-fold serial dilutions was additionally confirmed using gel agarose electrophoresis. For this purpose, a 2% gel was prepared with agarose powder (Universal, VWR International GmbH, Darmstadt, Germany) and GelRedTM (Biotium, Eching, Germany) as staining agent. Gel electrophoresis was performed at 5 volts/cm for 90 minutes to observe the characteristic ladder-like pattern of the LAMP products. After electrophoresis, the DNA was visualised and documented with gel doc (Gel Doc EZ Imager, Bio-Rad Laboratories GmbH, Munich, Germany). The obtained results were compared with DNA ladders of a marker (Biozyme) including DNA band sizes from 2000 bp to 50 bp (Quantitas, Biozym Scientific GmbH, Hessisch Oldendorf, Germany).

Results

Analytical specificity and sensitivity of the LAMP assay

The specificity of the LAMP assay was tested using 18 samples of the target species Thunnus albacares and 18 fish samples from five different non-target species (Table 2). All Thunnus albacares samples were amplified, while no amplification occurred when DNA templates from the 18 other fish species were used. Melting curve analysis by Genie II® showed specific melting temperatures of 85.4 ± 0.8°C for all Thunnus albacares samples (Fig 1). The results of LAMP and PCR were fully compliant.

Table 2

Analytical specificity of the cytB-LAMP assay for target and non-target species compared to real-time PCR.

Sample no.	Sample ID	Species	LAMP Amplification (mm:ss)	LAMP Melting (C°)	Real-time PCR
1	T3	Thunnus albacares	7:45	85.5	+
2	T4	Thunnus albacares	7:00	85.4	+
3	T5	Thunnus albacares	7:45	85.5	+
4	T6	Thunnus albacares	7:30	85.3	+
5	T7	Thunnus albacares	7:15	85.4	+
6	T8	Thunnus albacares	7:30	85.3	+
7	T25	Thunnus albacares	8:45	85.4	+
8	T26	Thunnus albacares	9:00	85.5	+
9	T27	Thunnus albacares	9:30	85.5	+
10	T28	Thunnus albacares	9:30	85.5	+
11	T29	Thunnus albacares	10:00	85.1	+
12	T30	Thunnus albacares	9:30	85.4	+
13	T31	Thunnus albacares	9:30	85.4	+
14	T32	Thunnus albacares	9:15	85.4	+
15	T34	Thunnus albacares	10:00	85.3	+
16	T35	Thunnus albacares	10:00	85.3	+
17	T37	Thunnus albacares	10:00	85.4	+
18	T38	Thunnus albacares	9:45	85.4	+
19	ANT1	Oncorhynchus keta	--	--	--
20	ANT2	Oncorhynchus keta	--	--	--
21	ANT3	Salmon salar	--	--	--
22	ANT4	Oncorhynchus nerka	--	--	--
23	ANT5	Gadus morhua	--	--	--
24	ANT6	Gadus morhua	--	--	--
25	ANT7	Gadus morhua	--	--	--
26	ANT8	Gadus morhua	--	--	--
27	ANT9	Gadus morhua	--	--	--
28	ANT10	Gadus chalcogrammus	--	--	--
29	ANT11	Gadus chalcogrammus	--	--	--
30	ANT12	Gadus chalcogrammus	--	--	--
31	T15	Katsuwonus pelamis	--	--	--
32	T16	Katsuwonus pelamis	--	--	--
33	T17	Katsuwonus pelamis	--	--	--
34	T18	Katsuwonus pelamis	--	--	--
35	T20	Katsuwonus pelamis	--	--	--
36	T23	Katsuwonus pelamis	--	--	--

Fig 1

Melting curves of LAMP products from various Thunnus albacares DNA samples.

The assay showed specific melting temperatures of 85.4 ± 0.8°C. T4PTC = positive control; NTC = non-template control.

The analytical sensitivity of the cytB LAMP assay was determined on the basis of 10-fold serial DNA dilutions. DNA amounts of 54 ng/μL to 0.54 pg/μL were successfully detectable in all three repetitions. Thus, the detection limit observed for Thunnus albacares was 0.54 pg/μL (Table 3, Fig 2).

Table 3

Analytical sensitivity of the LAMP assay using serial dilutions of Thunnus albacares DNA.

Dilution	10−0	10−1	10−2	10−3	10−5	10−5	10−6
Total DNA amount	(54 ng/μL)	(5.4 ng/μL)	(0.54 ng/μL)	(54 pg/μL)	(5.4 pg/μL)	(0.54 pg/μL)	(54 fg/μL)
Amplification time
1st run	7:15	8:00	9:00	10:15	11:45	14:30	-
2nd run	7:30	8:15	9:30	10.45	12:15	13:15	-
3rd run	6:45	7:30	8:45	9:45	11:45	13:45	-
Mean	6.97	7.82	8.92	10.1	11.68	13.63	-
SD±	0.45	0.45	0.43	0.51	0.41	0.60	-

Fig 2

Amplification profile of the Thunnus albacares LAMP assay using different dilutions.

The samples contained DNA concentrations of 54ng/μL to 54 fg/μL. NTC = non-template control.

Direct detection using MSwab

To avoid time-consuming DNA extraction, a fast and simple sample preparation method using the MSwab kit was performed. Amplification was observed in all swab samples from Thunnus albacares with detection times between 13 and 17 minutes (Fig 3) and specific melting temperatures of 85.4 ± 0.8°C.

Fig 3

Amplification profile of the Thunnus albacares LAMP assay using MSwab extraction of DNA.

T-SWAB = DNA from Thunnus albacares extracted by MSwab method; T4PTC = positive control; SAL1 = DNA from Salmon salar (negative extraction control); NTC = non-template control.

Detection by gel electrophoresis

Gel electrophoresis was performed with LAMP products obtained after amplification of the 10-fold serially diluted tuna reference DNA samples used for determining analytical sensitivity. Identical ladder-like DNA bands of all LAMP products were clearly visible after the run, indicating that all amplicons were specifically amplified (Fig 4).

Fig 4

Confirmation of the specificity of LAMP products obtained from 10-fold serially diluted tuna DNA samples using gel electrophoresis.

All samples showed an identical ladder-like band profile.

Discussion

Proper identification of fish species is very important for public health as well as for prevention of food fraud. The health risks associated with fish fraud are exposure to allergens, consumption of poisonous fish, accumulation of toxic metals, diarrhoea and sometimes death [19, 20]. Tuna consumption sometimes causes “scombroid poisoning” [20]. Such incidents indicate severe risks associated with mislabelling and species substitution of fish which also hampers consumer trust in the market.

Due to growing economic value, species substitution in food products is widespread, leading to health risks and economic losses. The European regulation (EC) 178/2002 requires full traceability of a product throughout the food chain and provides a legal basis for combatting food fraud. In accordance with the European regulation (EC) 1169/2011, all types of meat products must be correctly labelled with proper nomenclature. More specifically, in accordance with EU regulation 1379/2013, seafood labelling requires the inclusion of commercial and scientific names and information about whether the item was deforested [21, 22].

There are several molecular methods available for fish species detection. Among them, LAMP has gained in popularity in recent decades and is one of the most widely researched methods for species identification [11, 23]. Thus, it has already been used to detect several fish species such as eel (Anguilla anguilla) [12], salmon (Oncorhynchus mykiss) [24], cod (Gadus morhua) [14] and skipjack tuna (Katsuwonus pelamis) [15]. LAMP was applied successfully in various contexts and appears to be a fast and efficient alternative to PCR in the case of food fraud monitoring due to its low equipment requirements, easy handling and ability to be used in field [11]. The present study describes a LAMP assay for detecting Thunnus albacares. Even though other methods are available for tuna species detection, such as an investigation by PCR-RFLP [25], species-specific conventional PCR or real-time PCR [17], those are time-consuming and elaborate. In contrast, the cytB gene-based LAMP assay provides a robust, sensitive and specific tool for fast on-site detection of Thunnus albacares in less than 15 minutes.

The most challenging task in establishing a LAMP assay is the design of species-specific primers [14]. Both nuclear DNA and mitochondrial DNA were successfully used for primer design in fish species identification [14, 26, 27]. The most targeted mitochondrial genes for species identification include cytB, 12S rRNA, 16S rRNA and the cytochrome c oxidase subunit I (COX1) [28]. The cytB gene proved to be a successful marker for identifying seafood species and resolving species phylogenies [29]. Recently, Kappel et al. (2017) demonstrated the feasibility of an Illumina MiSeq NGS method targeting two short fragments of the mitochondrial cytB gene to authenticate tuna samples containing mixtures of species [30]. Another study in 2017 reported that the cytB mini-barcode was more competent than COI in the distinction of four Gadidae species (G. morhua, G. macrocephalus, Theragra chalcogramma, Pollachius virens) using HRM analysis [31]. There are other studies where cytB was successfully used for tuna species detection [32-34]. It was also a preferred target region for the development of LAMP assays detecting several fish and other animal species [12, 15, 16].

In this study, all Thunnus albacares samples collected from different supermarkets were correctly amplified using the newly established cytB LAMP assay. None of the other fish species used for control purposes displayed any cross-reaction. The high specificity of the LAMP assay for the authentication of Thunnus albacares was ensured by suitable LAMP primers that covered sufficient mismatches within the corresponding target regions of other fish species [14]. Due to a high degree of homology between the cytB sequences of the closely related tuna species Thunnus alalunga, Thunnus thynnus and Thunnus obesus, cross-reactions with the current LAMP primers might occur. However, this was not tested for in the present study, as the effect can be neglected due to the low European market dominance of these tuna species. In local supermarkets, generally only yellowfin and skipjack tuna products are available.

Other LAMP studies on detection of fish species showed varying degrees of analytical sensitivity. Very sensitive assays were able to detect up to 5 fg of skipjack tuna [35]. In another LAMP assay, 50 pg of skipjack tuna was detectable [15]. Further LAMP assays detected up to 500 pg of eel (genus Anguilla) [12], 285 pg of Atlantic cod (Gadus Morhua) [36], 37 pg of Pacific cod (Gadus macrocephalus) [36] and 197 pg of haddock (Melanogrammus aeglefinus) [36]. Compared to these results, the LAMP assay described in this study showed a high analytical sensitivity of 0.54 pg/μL. It enabled one of the main advantages of the established cytB LAMP assay, namely the simple and direct detection of Thunnus albacares in steak & fillet products without using any kit-based DNA extraction process. While kit extraction requires at least one time-consuming incubation step for accurate sample lysis as well as several washing steps and elution, dabbing samples with MSwab allowed direct detection of the target species with only marginally longer detection times compared to the kit-extracted DNA (Fig 3).

The LAMP assay was robust against the presence of PCR inhibitor substances like salt, spices and oil as it was able to detect DNA from various processed and canned products [37]. The short amplification time and low susceptibility towards inhibitors compared with PCR are noteworthy properties, enabling direct and reliable investigation of food samples in restaurants and of canned products [37, 38].

Overall, the cytB LAMP assay provided an accurate identification of the target species within 20 minutes using portable equipment. The application of the newly established cytB LAMP in food analysis will enable carrying out cost-effective and easy-to-perform tests in the field.

Conclusion

The LAMP assay described in this study provides a rapid, easy-to-perform, cost-effective and reliable method for identifying Thunnus albacares in tuna steaks, fillets or canned products. Highly specific primers based on the cytB gene allow for selective detection of the target species, avoiding cross-reaction with relevant non-target types of fish on the European market. Without the necessity for a complex DNA extraction process, the cytB LAMP assay can directly identify Thunnus albacares-containing products in less than 20 minutes. It may significantly help in detecting mislabelled products and cases of food fraud, and could therefore potentially be used by food monitoring authorities in retail, import trade surveillance, processing plants, restaurants or communal catering facilities.

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Reviewer #1: The manuscript is about the development of loop-mediated isothermal amplification (LAMP) assay for a fast and reliable identification of yellowfin tuna (Thunnus albacares) in commercial fish products. The topic is consistent with the scope of the journal and the manuscript is generally well structured. However, there are so many inaccuracies in the text and methodology, several changes in typos and spelling are needed and some information appear incomplete.

The text appears descriptive but lacks appropriate genetic studies and analysis.

For all these reasons, in my opinion, the paper should be thoroughly revised and is not suitable for publication in its current form.

Specific suggestions

Line 42: the Council Regulation (EEC) No 1536/92 defines “Tuna” all the species belonging to the genus Thunnus, please, better specify this concept.

Line 42: did you mean Thunnus thynnus? Please replace Thunnus thunus

Line 42: replace “Bluefin tuna” with “bluefin tuna”

Line 43: please, replace Thunnus alalanga with Thunnus alalunga

Line 45: please replace “omega 3 fatty acid” with “omega-3 fatty acid”

Line 54: please remove “which is”

Line 58: please replace “big eye tuna” with “bigeye tuna”

Line 58: please, cite also the bluefin tuna, that currently is the most expensive species

Line 60: some sentence about frauds for tuna species substitution, and some related reference, needs to be added. Further, about “variation in prices and tax imposed by the European Union on imported products” add some reference or information about imported products numbers

Line 63: also, proteomics analysis can be applied for tuna species identification in raw products, please cite them and add references

Line 70: please try to change the sentence by avoiding the use of the word “which” too many times

Line 74: The sentence “need longer time to pursue” give an incomplete information, since using specie-specific primers timing is very short. Thus, could be better to specify this concept and highlight the needs to design specie-specific reactions to shorten time analysis

Line 80: please, replace “cytochrome b” with “cytochrome-b”

Line 82: please, replace “food” with “fish”, since you only cited papers related to this food category

Line 90: specify how many steaks/canned samples you evaluated

Line 93: please replace “done” with “performed”.

Line 101: please replace “cytochrome b” with “cytochrome-b”.

Line 104: please, better specify what do you mean when you speak about “several target species”. The target species should be one (Thunnus albacares). Thus, do you mean several sequences of different specimens of the target species obtained from NCBI? Please, better explain the procedure.

Line 94-97: please delete this sentence. If you didn’t modify procedure, it is not necessary to explain kit’s instructions.

Line 107: which “closely related non-target species” did you considered? The table 2 reports other species, but they are not closely related to Thunnus albacares

Line 135: you used a ladder from 2000 bp to 50 bp, please specify the bp for your expected amplicons

Line 136: please, add a reference for “Direct Detection by MSwab” method

Line 152: you tested the specificity of the LAMP assay on your 18 samples. Did you use an internal control (COI, 16S, ecc) to be sure that your samples were exactly Thunnus albacares? Since some tuna species (T. obesus, albacares, alalunga, ecc.) may be different just for point mutation and produce the same Melting T°.

Line 152: you used 18 samples of the target species Thunnus albacares, plase specify it

Line 153: The “other fish species (non-target species)” listed in table 2 are not 18, you have 18 samples but only 5 “other” species.

Line 160: please replace “tenfold” with “ten-fold”.

Line 176: please replace “tenfold” with “ten-fold”.

Line 191: please replace “meat products” with “food products”

Line 197: please replace the sentence with “plants (25, 26), animals (20, 27-30), bacteria (31, 32), and herbal medicine.”

Line 201: please add the spot at the end of the sentence.

Line 206: please add the space after “LAMP”.

Line 208: please replace “species specific primers” with “species-specific primers”.

Line 240: please delete the repetition of the word “assay”.

Fig 4: Please, specify the bp for your amplicons.

Reviewer #2: 1.This work needs a deep review of English throughout the document.

2.In this study, the authors describe an assay for yellowfin tuna with a lower limit of detection of 540 fg/μl and good specificity as well as providing a rapid DNA extraction method.

3.In my opinion, the authors needed to benchmark a recognized assay for 36 samples, and fluorescent quantitative PCR（qPCR） was a good choice.

4. LAMP as a common method for isothermal amplification needs to be compared with other isothermal amplification methods such as RPA, RCA, CPA, SDA, and HDA, and the authors need to add this section in the Introduction and Discussion section.

5.The authors mentioned that the detection target sequences were compared, and the results of this section need to be presented as supplementary material.

6.Changes need to be made to Figure 4, and I think the left Marker is best deleted.

Reviewer #3: A rapid method for yellowfin tuna (Thunnus albacares) authentication by LAMP was developed in the present work. Generally, I would say it is an interesting work, and the potential of LAMP in seafood species identification has been verified. However, there are lots of problems with the present one:

1) the results should be carefully described,

2) the figures and tables should be detailedly explained

3) the comparison between traditional method and rapid extraction method should be described and explained

4) besides gel electrophoresis, visual detection showed more advantages, which are not used in the present work.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Submitted filename: Review comments.docx

Click here for additional data file.

15 Aug 2022

The authors would like to thank the reviewers for the constructive comments which helped to improve this manuscript. In the rebuttal letter the authors refer to all comments and hope to have addressed all aspects adequately.

19 Sep 2022

Development of loop-mediated isothermal amplification (LAMP) assay for rapid and direct screening of yellowfin tuna (Thunnus albacares) in commercial fish products

PONE-D-22-09199R1

Dear Dr. Abdulmawjood,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: No

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: No

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: No

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: No

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: I agree to this manuscript for publication on Plos one，I agree to this manuscript for publication on Plos one.

Reviewer #3: The Author response is missing, and the revised manuscript still has lots of errors.

The results section and the figures/tables, as suggested in my previous comment, should be carefully described.

The present one is not acceptable.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #2: No

Reviewer #3: No

**********

20 Sep 2022

PONE-D-22-09199R1

Development of loop-mediated isothermal amplification (LAMP) assay for rapid and direct screening of yellowfin tuna (Thunnus albacares) in commercial fish products

Dear Dr. Abdulmawjood:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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