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

1

Highly simple and sensitive molecular amplification-integrated

2

fluorescence anisotropy for rapid and on-site identification of

3

adulterated beef

4

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,

7

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

12

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

13

FP measurement a highly attractive alternative sensing technology to conventional

14

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

15

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

16

molecular weight of fluorescent labeled primers increased rapidly by slight

17

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

18

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

19

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

20

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

21

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

22

were successfully authenticated with satisfied results.

23

∗

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,


2-4

immunoassays,9

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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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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


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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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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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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3.3 Optimization of primer amounts 9 ACS Paragon Plus Environment


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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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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

260

rapid

261

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

262

adulterated duck meat component was rapidly measured without complicated

263

purification and separation procedures in the whole detection process. Authentication

264

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

265

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

266

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

authentication

of

adulterated

beef

samples


in

the

easy

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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

275

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

276

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,

281

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

282

Notes

283

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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validation of a TaqMan real-time PCR assay for the identification and quantification

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fluorescence anisotropy probe for DNA-based bioassays: The case of SYBR Green.

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Biosens. Bioelectron. 2017, 90, 140-145.

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and proflavine-probed fluorescence polarization biosensor for ligand-RNA interaction

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assay. Sensor. Actuat. B-C. 2018, 257, 666-671.

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for ATP using aptamer as affinity ligand and dye-labeled ATP as fluorescence tracer.

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Tetramethylrhodamine-Labeled

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Fluorescence Anisotropy Detection of a Small Molecule, Ochratoxin A. Anal. Chem.

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Sensing

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Aptamer

for

Chem.

Noncompetitive


2018,

doi.

Aptamer-Based

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milk. Anal. Chem. 2017, 89, 5900-5908.

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Optimization of the structure-switching aptamer-based fluorescence polarization assay

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405



406 407

Captions of Figures

408

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

409

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

410

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

411

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

412

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

413

value and adulteration rate.

414

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

415

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

416

Negative control.

417


Page 18 of 27

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418 419

420 421 422

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



423

424 425 426

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

427

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

428

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

429

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

430

value and adulteration rate.


Page 20 of 27

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431

432 433

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

434

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

435

Negative control.

436



Page 22 of 27

437

Supporting Information

438

Highly simple and sensitive molecular amplification-integrated

439

fluorescence anisotropy for rapid and on-site identification of

440

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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446

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

447

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

448

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

449

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

450

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

451

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

454

fluorescence intensity of vertical light.

455

݉ܲ = 1000

ூ∥ ିூ఼ ூ∥ ାூ఼

(1)

456 457



Page 24 of 27

458 459

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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465 466

467 468 469

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

470

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

472

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

473

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

474

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

475

consistent with A.

476



477

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.


Page 26 of 27

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487

Reference

488

(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

490

Food Safety 2016, 36, 367-374.

491

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

492

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

493

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

494

392-398.

495 496 497


Highly Simple and Sensitive Molecular Amplification-Integrated

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