Highly simple and sensitive molecular amplification-integrated

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Highly simple and sensitive molecular amplification-integrated fluorescence anisotropy for rapid and on-site identification of adulterated beef Dongqing Qiao, Jianguo Xu, Panzhu Qin, Li Yao, Jian-feng Lu, Sergei Eremin, and Wei Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01374 • Publication Date (Web): 18 May 2018 Downloaded from http://pubs.acs.org on May 18, 2018

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Analytical Chemistry

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Highly simple and sensitive molecular amplification-integrated

2

fluorescence anisotropy for rapid and on-site identification of

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adulterated beef

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Dongqing Qiao , Jianguo Xu , Panzhu Qin , Li Yao , Jianfeng Lu , Sergei Eremin#, Wei Chen











†,∗

5 6



School of Food Science & Engineering, Engineering Research Center of Bio-process, MOE,

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Hefei University of Technology, Hefei 230009, China

8

#

National Research Technical University "MISiS", Leninsky Prospekt 4, Moscow, Russia

9 10

Abstract

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Fluorescence polarization (FP) signal is a self-referencing fluorescence signal, and it

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is less dependent on dye concentration and environmental interferences, which makes

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FP measurement a highly attractive alternative sensing technology to conventional

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fluorescent detection methods. Here we adopted a strategy for rapid increase of

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molecular weight to increase the FP signal for the detection of meat adulteration. The

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molecular weight of fluorescent labeled primers increased rapidly by slight

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pre-amplification and FP value were varied accordingly. We found a positive

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correlation between adulteration ratio and the FP signals. Detection limit for

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adulterated beef can be reached as low as 0.1% (wt.%), meeting or better than the

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most detection requirements. On the basis of this proposed amplification-integrated

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FP method, both the standard samples and the commercial processed beef samples

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were successfully authenticated with satisfied results.

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Corresponding author: W. Chen, e-mail: [email protected], Researcher ID: F-4557-2010,

ORCID: 0000-0003-3763-1183 (W. C.)

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1. Introduction

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Meat products account for a large proportion of food consumptions of the residents all

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over the world. However, current situations of meat quality in China and other

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countries is worrying.1 Low-priced pork, duck or other meat from dead animals have

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been adulterated in beef or mutton of higher price, which have been frequently

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exposed and reported, such as the “horsemeat scandal” in 2013. Nowadays,

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adulteration of meat products has become a very serious problem which greatly limits

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the development of food industry and affects the public health and safety, religious

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factors of meat choice and unfair competitions in the commercial market.

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Therefore, to protect the consumer rights, public safety of meat-related disease

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transmission and normal commercial rules of food market, it is critically important to

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develop a reliable, rapid and efficient method for easy and accurate authentication of

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meat adulteration.5,6

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Previous authentication studies of adulterated meats focus on the adoption of

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characteristic proteins or nucleic acids as the target analytes.7,8 For protein analysis,

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the

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chromatography,10 mass spectrometry (MS) and spectroscopy.11,12 Great achievements

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have been reached for authentication of adulterated meat with these methods.

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However, due to the intrinsic properties of proteins, processed meat foods cannot be

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detected with abovementioned protein-based methods because of the denaturation of

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proteins without considering the cost of the equipment.13,14 Besides, for

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immunoassays, the antibodies against the specific proteins should be additionally

widely

reported

methods

include

electrophoresis,

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immunoassays,9

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Analytical Chemistry

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prepared for detection. Alternatively, methods taking genetic materials as the target

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analytes have been extensively studied to overcome the limitations of protein-based

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methods.15 And the current “gold-standard” protocol for authentication of adulterated

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meat is also the DNA-based real-time polymerase chain reaction (RT-PCR). This

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gold-standard protocol of RT-PCR can realize the detection of adulterated meat with

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satisfied specificity and sensitivity for both the raw and processed meat samples.16,17

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Excellent detection performance of this gold-standard protocol can be attributed to the

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amplification ability of PCR.18-20 However, the high price of RT-PCR and highly

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trained personnel determine that it cannot be applied for routine and on-site screening

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or quantitative authentication of meat adulteration. Meanwhile, professional

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experience is also stringently required for TaqMan probe design of RT-PCR. In order

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to avoid the cumbersome post-treatments of normal PCR and expensive fluorescent

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module of RT-PCR, a simple and rapid strategy with low cost is required for

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integration with conventional PCR for on-site and routine authentication of meat

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adulteration with high efficiency.

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Interestingly, fluorescent polarization (FP) is a potential candidate for rapid and easy

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measurement of fluorescent signals.21 FP measurement is based on the principle of

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self-referencing fluorescence sensing technology. In detail, the FP intensity is

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determined by both the parallel and perpendicular fluorescent intensities excited by

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the vertical-polarized light rather than the dye concentration or environmental

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interference.

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This property of FP makes it much more attractive than other fluorescent signal 3 ACS Paragon Plus Environment

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read-out protocols.22,

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one-step direct “add-and-measure” model without additional separation or remove of

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excess reagents or biomolecules, which also makes it highly suitable for real-time and

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routine monitoring of the dynamic changes.24-31

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Hence, in this research, we present a novel amplification-integrated FP detection

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technique for accurate and specific authentication of duck meat adulterated beef

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samples based on the designed functional primer set for amplification. The

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amplification induced molecular weight variations of functional primers influence the

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FP values for authentication. Both the raw and processed beef samples with different

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adulterated ratios are rapidly and successfully authenticated qualitatively and

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quantitatively with satisfied results.

Besides, of great significance, FP measurement is in a

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2. Experimental

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2.1 Chemicals and apparatus

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FP signal was measured on a portable Sentry 201 (Milwaukee, USA). Taq polymerase

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and dNTP were purchased from Sangon Biotech (Shanghai, China). The 4S Red Plus

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dye was also ordered from Sangon Biotech (Shanghai, China). The conventional

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primers were synthesized from General Biosystems (Anhui, China). The functional

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group functionalized primers were also prepared and ordered directly from this

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company (the detailed chemical structures of FITC-labeled primers were

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demonstrated in Figure S2 in SI). The detailed sequences of all primers were showed

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in Table S1. Of note, three types of gene markers of duck meat were adopted in this 4 ACS Paragon Plus Environment

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Analytical Chemistry

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research including 16S rRNA, Cyt b and ND1 for the amplification templates of 80,

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204 and 322 bp, respectively. Sodium dodecyl sulfate (SDS), polyethylene glycol

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20000

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Tris(hydroxymethyl)aminomethane, proteinase K solution (20 mg/mL), sodium

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hydrogen phosphate and sodium dihydrogen phosphate were all purchased from

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Sinopharm Chemical Reagent Co., Ltd. (Wuhan, China) of analytical grade and used

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directly without any further purification. High-purity deionized water (>18 MΩ) was

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used throughout the research.

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2.2 Samples preparation

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Fresh and raw beef and duck samples were purchased from local supermarkets and

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stored at -20 °C prior to analysis. For raw meat sample analysis, beef and duck meat

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samples at different weight ratios were mixed and crushed together completely.

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Different adulteration ratio (0%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 50% and

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100% (w/w)) of beef samples were prepared. After drying of the crushed samples, the

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powder samples were ready for extraction of DNA; For processed meat sample

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analysis, after mixing and crushing of meat samples, the crushed mixtures were

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cooked with steamer. Afterward, the boiled meat samples were also ready for

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following DNA extractions.

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2.3 DNA extractions of the meat samples

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The genomic materials were extracted and purified by the magnetic nanoparticle

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(MNP)-based protocol developed by our lab with minor modifications.32-34 Briefly

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and firstly, 700 µL SDS buffer (Tris-HCl 20 mM, 1 % SDS, EDTA 5 mM and NaCl

(PEG-20000),

ethylene

diamine

tetra-acetic

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acid

(EDTA),

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400 mM, pH 8.0) at 55 °C and proteinase K (35 µL) were added to the dried meat

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powder (50 mg) and incubated at 55 °C for 2 h with vigorous vortexing. Then, the

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mixture was centrifuged at 12 000 g for 10 min and the supernatant was transferred to

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a new tube containing PEG ⁄ NaCl solution (30 % PEG and 2 M NaCl). MNPs were

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added to different portions of this solution at the same concentration to separate and

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purify the DNA samples. After 10 min, the MNPs were collected under an applied

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magnetic field and rinsed three times with ethanol to elute the genomic DNA in TE

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buffer. The extracted DNA was confirmed by agarose electrophoresis and the results

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were further analyzed by PCR.

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2.4 Amplification conditions

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Detailed conditions adopted for PCR were demonstrated in SI. Negative control

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reaction mixtures contained sterile distilled water instead of the extracted template

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DNA. Pure distilled water was adopted as the blank control. 5 µL of each PCR

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product was confirmed by electrophoresis in 2% (w/v) agarose in 1 ×TBE containing

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4S Red Plus ordered from Sangon Biotech (Shanghai, China). Finally, the PCR

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products were collected and ready for measurement with portable FP device. Of note,

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the amplification cycle was also optimized to achieve the best detection performance

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in the comparable short period.

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2.5 Fluorescence polarization assay of the adulterated beef samples

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Amplified products were added directly into 10 mM phosphate buffer solution (PB,

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pH 7.4) and mixed well. After that, FP measurement was immediately performed

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using portable Sentry 201 and the FP values were obtained based on the equation 6 ACS Paragon Plus Environment

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Analytical Chemistry

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demonstrated in SI.

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3 Results and discussion

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3.1 Mechanism for rapid quantification of adulterated beef samples

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(Scheme 1)

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In a specific system, the FP value of the fluorescent labeled molecule (FITC labeled

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primer) is sensitive to the changes in rotational motion of the probe itself. Before the

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amplification, the fluorescent labeled primer is comparable free in the solution and

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rotates fast, showing a relative small FP value. In the presence of adulterated duck

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meat component in the beef samples, the amplification will occur and the molecular

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weight of the fluorescent labeled primer will be increased dramatically, which will

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decrease the rotation rate of the fluorescent probe and increase the FP value

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accordingly. The variation of the FP value is in proportion to the original amount of

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adulterated duck meat in beef samples and the adulterated duck meat can be

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determined based on the variations of FP signal. Previously, molecular weight

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increased measurements including structure-switching,35 mass-augmented and

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target-induced displacement have been developed for quantitative analysis of target

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analytes.36, 37 Different from the previous reports, a special molecular weight increase

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strategy is designed by taking advantage of the PCR amplification in this research.

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The fluorescein isothiocyanate (FITC, called tracer) labeled primer is designed as the

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fluorescent reporter in FP detection. In the absence of template, the molecular weight 7 ACS Paragon Plus Environment

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of the tracer is small, and it rotates fast in the solution showing small FP value.

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However, in the presence of target adulterated duck meat component, the tracer will

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be amplified to form long double-stranded DNA products, resulting in obvious

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increase of FP value (Scheme 1). The variation of FP value can accurately reflect the

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ratio of adulterated components in beef samples and rapid and sensitive authentication

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of adulterated components in beef samples can be realized as demonstrated in Scheme

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1.

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3.2 Optimization of the primer design and selection

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The selection of primers is a critical factor in this research due to the fact that both the

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amplification efficiency and amplification length can influence the variation of the FP

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value. Therefore, two aspects should be considered for achieving the best detection

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results. For one thing, some primer set can realize the amplification of target analyte

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with the presence of dimer, which is not crucial for the results distinguish of

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molecular based methods. However, for FP-based determination, the presence of

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dimer can also induce the change of FP value, which can be treated as the background

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signal and sacrifice the sensitivity of the method; for the other, the amplification

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length can affect the final FP value greatly. While the amplification length is too short,

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the variation of FP value will not be distinguished from the tracer itself due to the

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limited increase of molecular weight. The detailed optimization results were shown in

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Figure S1. In Figure S1a, it is easy to find that primer set 2 and 3 have the observable

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presence of dimer of negative controls in the gel results. Meanwhile, for the FP value, 8 ACS Paragon Plus Environment

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Analytical Chemistry

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although the highest FP value is obtained of primer set 3, its background signal of

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negative control is also the highest. Comparatively, primer set 1 is the best choice for

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research. Of note, for primer set 1, 2 and 3, the length of the amplification product is

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80 bp, 204 pb and 322 bp, respectively. Theoretically, the primer set 3 with the longest

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amplification length should be adopted for the increase of molecular weight. In order

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to further confirm the optimization, two new primer sets of primer 1-L1 and primer

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1-L2 were designed based on the sequence of primer 1 (ten-based extension with

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different sequence of original primer 1 at 5’ end with italic font and underlined,

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primer1-L1: 5’-aag cct tcct aag cct tcc tct agc tcagc-3’ and primer1-L2: 5’-ccg tcc taa

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t aag cct tcc tct agc tcagc-3’). From the results in Figure S1b, it is easy to find that the

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new designed primer 1-L1 has the highest FP value followed by primer 1 and primer

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1-L2. For comparison between primer 1-L1 and primer 1, the extended ten-base

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contributes to the molecular weight of the products and induces the increase of FP; for

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comparison between primer 1-L1 and primer 1-L2, the same length of both

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amplification length and extended base number while the different base adjacent to

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the labeling fluorescein, the FP value of primer 1-L1 is higher than that of primer

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1-L2. The reason for this phenomenon may be that the labeled fluorophore in the

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primer has the certain interaction with the adjacent bases or the molecular tumbling of

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the fluorescein, which limiting the free swing of the fluorophore and corresponding

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FP signal variations 27.

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With the selected primer set 1, the concentrations were further optimized to get the

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best signal/noise ratio. From results demonstrated in Figure 1a, the increase of

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concentration of primer set 1 can greatly improve the positive signal variation without

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increase of negative groups till 60 nM. Further increase of primer concentration, the

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signal of positive groups comes to a stable zone while that of the negative groups

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increases a little. Therefore, 60 nM is selected as the optimal concentration for further

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research.

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3.4 Optimization of the amplification efficiency

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The FP signal is changed by the amplification induced molecular weight variations

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and it is easy to understand that the amplification directly determines the final

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detection performance. Optimization of amplification cycle, two aspects should be

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considered. Firstly, to insure enough FP signal variations for determination, complete

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amplification should be guaranteed; secondly, excessive amplification will induce the

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presence of dimer and non-specific amplification products, which will induce the

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increase of background signal (noise signal). The optimization results are shown in

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Figure 1b and it is obvious that the increase of cycle number produces the stronger FP

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signal for determination. Meanwhile, it is also noted that at 35 cycles, the background

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signal is also enhanced greatly (results not shown due to the log treatment). Therefore,

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considering both the detection efficiency and authentication performance, 25-cycle is

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adopted as the best amplification condition for detection. Of note, this 25-cycle

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amplification is not enough for routine PCR or RT-PCR based detections. The 10 ACS Paragon Plus Environment

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Analytical Chemistry

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FP-integrated signal report protocol can improve the detection efficiency at shorter

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time.

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(Figure 1)

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3.5 Detection performance of the adulterated beef samples

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Under the above optimized conditions including samples DNA extracted with

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magnetic beads, amplified with primer set 1 at 60 nM and amplification cycles of 25,

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fluorescence polarization detection of adulterated duck meat in the beef samples were

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carried out. When the beef samples with different adulteration ratio were measured

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directly with Sentry 201 and the corresponding FP values were analyzed against the

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adulteration ratio as shown in Figure 1c and d. It is quite obvious that the FP signal

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increases with the increase of adulteration ratio of duck meat in beef samples and as

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low as 0.1% adulteration ratio can be well distinguished from the negative control.

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Furthermore, the calibration curve for quantitative analysis was constructed with the

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R square of 0.9979 and the detection limit was calculated to be 0.36% of adulterated

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ratio (Figure 1d). From the achieved results, it can come the conclusion that the

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detection limit of this FP based method is comparable to that of the classic RT-PCR

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while the whole detection time is a little shorter with only 25-cycle amplification

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required. Therefore, integration of FP with the PCR can attribute to the improvement

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of the authentication efficiency of adulterated meat.

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(Figure 2)

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3.6 Practical application of FP detection of adulterated duck meat component in processed beef products

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Finally, the developed FP detection protocol was further applied for authentication of

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adulterated duck meat component in commercial processed beef products. All treated

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samples were determined with the developed method the direct measured FP signals

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were all shown in Figure 2. Pure raw beef sample was determined as taken as the

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negative control (sample 19 in Figure 4). From the results demonstrated in Figure 2, it

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is obvious that two of eighteen processed beef samples were adulterated with duck

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meat (signal higher than 110% of negative control was treated as positive samples),

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which were consistent with those of the RT-PCR method.

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4 Conclusions

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In summary, a simple fluorescence polarization detection method was developed for

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rapid

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“amplification-add-measure” model. After the common and shorten amplification,

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adulterated duck meat component was rapidly measured without complicated

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purification and separation procedures in the whole detection process. Authentication

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of adulterated duck meat component was achieved by the variation of the fluorescence

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polarization signal with the detection limit as low as 0.1% and 0.36% for qualitative

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and quantitative analysis, respectively. Furthermore, the proposed strategy can also be

authentication

of

adulterated

beef

samples

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in

the

easy

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Analytical Chemistry

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expected to provide a universal platform for authentication of adulterated meat with

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proper screening of primer set. Meanwhile, this strategy will further widen the

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application of fluorescence polarization-based detection methods by integration with

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classic and conventional PCR protocol.

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ASSOCIATED CONTENT

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Supporting Information

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The supporting information including the sequence of the functional primers, the

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conditions of the amplification and the optimization results is available free of charge

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on the ACS Publications website.

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AUTHOR INFORMATION

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Corresponding Author

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* e-mail: [email protected], Researcher ID: F-4557-2010,

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ORCID: 0000-0003-3763-1183 (W. C.)

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Notes

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The authors declare no competing financial interest.

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Acknowledgement

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This work is financially supported by the grant of 2017YFF0208600, China

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Agriculture Research System-48 (CARS-48), the special fund of central university

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2017HGPA0162, PA2017GDQT0018, NSFC 21475030 and the S& T Research 13 ACS Paragon Plus Environment

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Project of Anhui Province 15czz03109.

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Penicillium nalgiovense, on Dry-Cured Sausage ‘Salchichón’ Using a SYBR

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Green-Based Real-Time PCR. Food Anal. Method 2015, 8, 1582-1590.

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406 407

Captions of Figures

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Scheme 1. Schematic illustration for the detection of adulteration by FP.

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Figure 1. (a) FP test results of the primers optimization. Red line represents positive (100%

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duck), black line represents negative (0% duck); (b) The cycle number optimization of adulterated

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meat. As illustrated in the figure, different colors represent different amplification cycles; (c) FP

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response of a series of standard adulterated meat samples. (d) Linear relationship between FP

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value and adulteration rate.

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Figure 2. Compare the amount of duck meat in different types of cooked meat samples.1-4: Beef

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granules. 5-8: Beef jerky. 9-12: Beef back strap. 13-15: Beef strip. 16-18: Beef paste. 19:

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Negative control.

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420 421 422

Scheme 1. Schematic illustration for the detection of adulteration by FP.

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424 425 426

Figure 1. (a) FP test results of the primers optimization. Red line represents positive (100%

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duck), black line represents negative (0% duck); (b) The cycle number optimization of adulterated

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meat. As illustrated in the figure, different colors represent different amplification cycles; (c) FP

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response of a series of standard adulterated meat samples. (d) Linear relationship between FP

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value and adulteration rate.

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Figure 2. Compare the amount of duck meat in different types of cooked meat samples.1-4: Beef

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granules. 5-8: Beef jerky. 9-12: Beef back strap. 13-15: Beef strip. 16-18: Beef paste. 19:

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Negative control.

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Supporting Information

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Highly simple and sensitive molecular amplification-integrated

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fluorescence anisotropy for rapid and on-site identification of

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adulterated beef

441











Dongqing Qiao , Jianguo Xu , Panzhu Qin , Li Yao , Jianfeng Lu , Sergei Eremin#, Wei Chen

†,∗

442 443 444



School of Food Science & Engineering, Hefei University of Technology, Hefei 230009, China

#

National Research Technical University "MISiS", Leninsky Prospekt 4, Moscow, Russia

445



Corresponding author: W. Chen, e-mail: [email protected], Researcher ID: F-4557-2010,

ORCID: 0000-0003-3763-1183 (W. C.)

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The detailed PCR condition for amplification: PCR reaction mixtures comprised the

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reaction buffer, dNTP (0.2 mM), primer (0.2 mM), 4 µL DNA, 0.1 U Taq DNA

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polymerase and deionized water to a final volume of 25 µL. PCR was carried out

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according to the following program: (i) 5 min at 94 °C; (ii) 25 cycles of denaturation

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for 30 s at 95 °C, annealing for 30 s at 52.3 °C, extension for 30 s at 72 °C; (iii) 3 min

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at 72 °C.

452

The FP value (mP, 1 P = 1000 mP) can be obtained according to the following

453

Equation (1), in which I∥ is the fluorescence intensity of parallel light, and I⊥ is the

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fluorescence intensity of vertical light.

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݉ܲ = 1000

ூ∥ ିூ఼ ூ∥ ାூ఼

(1)

456 457

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Table S1. The detailed sequence information of all primers utilized in this

460

research Primer P1-F-FITC

Gene marker 16S Rrna S1

P1-R P2-F-FITC

Cyt b S1

P2-R P3-F-FITC

ND1 S2

P3-R

461 462 463

P1-L1-F-FITC

16S rRNA

P1-L2-F-FITC

16S rRNA

Sequence 5’-aag cct tcc tct agc tca gc-3’ 5’-aga aaa tgc ttt agt taa gtc-3’ 5’-ccg tcc taa tcc tat tcc tgg tc -3’ 5’-gga ata gga gga tgg tga agt aag ta-3’ 5’-gcc aca aac aac aat agt aag c-3’ 5’-ccc gag gtt cag gtc tac ta-3’ 5’-aag cct tcc t aag cct tcc tct agc tca gc-3’ # 5’-ccg tcc taa t aag cct tcc § tct agc tca gc-3’

#

Product Size/bp

80

204

322

80 80

P1-L1-F-FITC, the same amplification primer sequence as P1-F-FITC with ten-base extension; P1-L2-F-FITC, the same amplification primer sequence as P1-F-FITC with ten-base extension and also the different extension sequence compared to P1-L1-F-FITC;

§

464

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467 468 469

Figure S1. The FP and electrophoresis comparison results of amplification with three

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types of designed primer sets. (a) The picture on the left is the FP test results for primer 1,

471

primer 2 and primer 3, respectively. Red represents positive (100% duck), gray represents negative

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(0% duck). The picture on the right is the result of the agarose gel electrophoresis. M: marker.

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Lane1: primer1 positive. Lane2: primer1 negative. Lane3: primer2 positive. Lane4: primer2

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negative. Lane5: primer3 positive. Lane6: primer3 negative. (b) Except the primers, Figure B are

475

consistent with A.

476

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478

479

480

481

482 483 484 485 486

Figure S2. The chemical structures of the FITC-labeled primers including P1-FITC (F), P2-FITC (F), P3-FITC (F), P1-L1-FITC (F) and P1-L2-FITC (F), F means forward primer.

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Reference

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(S1) Qin, P.; Hong, Y.; Kim, H. Multiplex ‐ PCR Assay for Simultaneous

489

Identification of Lamb, Beef and Duck in Raw and Heat‐Treated Meat Mixtures. J

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Food Safety 2016, 36, 367-374.

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(S2) He, H.; Hong, X.; Feng, Y.; Wang, Y. S.; Ying, J.; Liu, Q.; Qian, Y. W.; Zhou, X.

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K.; Wang, D. S. Application of Quadruple Multiplex PCR Detection for Beef, Duck,

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Mutton and Pork in Mixed Meat. Journal of Food & Nutrition Research 2015, 3,

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392-398.

495 496 497

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