Research Progress on Energy Plants in Piggery Wastewater Treatment

Ziqiang AO Guiqun PENG Cheng JIANG Bing YAN Jihai XIONG Jiaqi FU

AbstractAiming at the market demand for rapid detection of tetracyclines, fluoroquinolones and sulfonamides in milk, a golloidal gold immunochromatography test strip for simultaneous detection of tetracyclines, fluoroquinolones and sulfonamides in milk was prepared based on the principle of competitive inhibition immunochromatography. The performance indicators of the test strip were verified. The results showed that the test strip can simultaneously detect 4 tetracyclines, 13 fluoroquinolones and 13 sulfonamides, and the detection limits all can meet the national residue limits; the tests strip exhibited false positive rate≤5% and false negative rate=0; and no crossreaction with other drugs was commonly found in milk, indicating good specificity. The method is simple, rapid, and has low cost and easy popularization. It provides a means for realizing onsite rapid detection and is of important practical significance to guarantee of safety of milk and dairy products in China.

Key wordsMilk; Tetracyclines; Fluoroquinolones; Sulfonamides; Colloidal gold immunochromatography assay



Received: July 7, 2018Accepted: October 9, 2018

Supported by Hebei Science and Technology Program (16275507D).

Yuping WAN (1982-), male, P. R. China, veterinarian, devoted to research about food safety rapid detection technology.

*Corresponding author.

In order to protect animal health, promote rapid animal growth, and improve feed conversion ratio, antibiotics are widely used, mainly including tetracyclines, fluoroquinolones, sulfonamides, peptide antibiotics, and βlactam antibiotics［1］. Among them, tetracyclines (TCs) are a class of broadspectrum antibiotics produced by Streptomyces, which are chemically related to derivatives of multiring tetraphenylcarboxyamide mother nucleus［2］; fluoroquinolones (FQs) are a class of synthetic antibacterial drugs containing a basic structure of 4quinolone, which inhibit bacterial DNA synthesis［3］, thereby realizing their antibacterial effect; and sulfonamides (SAs) are a class of chemotherapeutic drugs with paminobenzenesulfonamide structure, which inhibit bacterial reproduction by affecting the synthesis of nuclear proteins［4］.

However, due to drug abuse and noncompliance with the withdrawal time and differences in animal metabolism, drug residues appear in animal foods and endanger human health. Especially, drug residues in meat, eggs and dairy products are the most serious problem that affects human health. Therefore, China has stipulated that the maximum residue limit of oxytetracycline, tetracycline and chlortetracycline in milk is 100 μg/L, and doxycycline should not be detected; and the maximum residue limits of fluoroquinolones in milk are as follows: enrofloxacin and ciprofloxacin 100 μg/L, danofloxacin 30 μg/L and flumequine 50 μg/L, and the maximum residue limit of nine sulfonamides such as sulfamethazine, sulfadiazine and sulfapyridine in milk is 100 μg/L［5］.

At present, the detection methods of TCs, FQs and SAs residues in animalderived foods mainly include microbiological method［6-7］, high performance liquid chromatography［8-9］, chromatographymass spectrometry［10-11］, highefficiency capillary electrophoresis［12］, immunoassay［13-14］ and so on. Due to the large difference in the nature of veterinary drugs, existing residue analysis generally establishes corresponding detection methods according to the drugs in the same family with similar chemical structures, while these analytical methods are mostly focused on the detection of single antibiotics, and there are few methods for simultaneous detection of multiple antibiotics, which could not meet the current increasing demand for veterinary drugs and the increasing analytical flux. At present, there also have been studies on the detection of multiple types of antibiotics by LCMS/MS［15-18］, but the method has the disadvantages of complex sample preparation process, high instrumentation, high cost and low analysis speed, which is only suitable for the confirmation analysis of samples, and not suitable for screening and onsite testing of large quantities of samples. In contrast, colloidal gold immunochromatography assay has short detection time, good stability, easy operation, and no need for other instruments and equipment, and the results are intuitive and reliable. It is suitable for onsite rapid screening of large quantities of samples and is of great significance to control of multiple antibiotic residues. Therefore, this study was conducted to establish a colloidal gold immunochromatography assay method that can simultaneously detect tetracyclines, fluoroquinolones and sulfonamides in milk, so as to meet the detection requirements of these three types of antibiotic residues in animalderived foods at home and abroad.

Materials and Methods

Materials and reagents

Milk: commercially available; artificial antigens and monoclonal antibodies of tetracyclines, artificial antigens and monoclonal antibodies of fluoroquinolones, and artificial antigens and monoclonal antibodies ofsulfonamides: Beijing Kwinbon Biotechnology Co., Ltd.; standards of tetracyclines, fluoroquinolones and sulfonamides (purity≥99%): Sigma; chemical reagents such as chloroauric acid, trisodium citrate, phosphoric acid and potassium carbonate: Beijing Chemical Reagent Company; goat antimouse immunoglobulin IgG: Golden Bridge International Inc.; nitrocellulose membrane, sample absorbent pad, absorbent pad: Shanghai Liangxin Technology Co., Ltd.

Instruments and equipment

UVmini1240 ultravioletvisible spectrophotometer: Shimadzu, Japan; Biodot filmforming apparatus: Changsha Boyou Biochemical Products Co., Ltd.; 902 thermostatic magnetic stirrer: Shanghai Zhenrong Scientific Instrument Co., Ltd.; DH1012 type electricheating constanttemperature incubator: Shanghai Yiheng Technology Co., Ltd.; S4800 cold field emission scanning electron microscope: Hitachi, Japan; CR21GIII highspeed refrigerated centrifuge: Hitachi, Japan.

Methods

(1) Preparation and quality analysis of colloidal gold

Colloidal gold was prepared by trisodium citrate reduction［19］. The prepared colloidal gold was detected by an ultravioletvisible spectrophotometer and an electron microscope.

(2) Selection of optimal antibody dosage for stabilizing colloidal gold

The optimal amount of antibody for stabilizing colloidal gold was determined by visual inspection. The pH of colloidal gold was adjusted to 7.2 with 0.1 mol/L K2CO3 solution, and the monoclonal antibody was gradually diluted to 5-40 μg/ml with 0.01 mol/L phosphate buffer (pH 7.4). The optimal amount was determined according to Table 1. The prepared colloidal gold was shaken uniformly, stood for 5 min, and added with 10% NaCl, followed by shaking uniformly and standing for 2 h.

Table 1Determination of the optimal dosage of antibody for stabilizing colloidal gold

No.Antibody∥μgColloidal gold∥ml10% NaCl∥μl

101.0100

251.0100

3101.0100

4151.0100

5201.0100

6251.0100

7301.0100

8351.0100

9401.00

Tube 1 is a control tube to which no antibody is added, and 0.1 ml of tridistilled water is added thereto; and tube 9 is a control tube to which no sodium chloride is added, and 0.1 ml of tridistilled water is added thereto.

(3) Preparation of colloidal goldlabeled monoclonal antibody

Under magnetic stirring, the pH of colloidal gold was adjusted to 7.2 with 0.1 mol/L K2CO3 solution. Corresponding monoclonal antibody was added to the colloidal gold solution at the optimal amount of antibody per milliliter of colloidal gold solution, followed by stirring and mixing for 10 min. 10% bovine serum albumin (BSA) was added to a final concentration of 1% in the colloidal gold solution, followed by standing for 10 min. The liquid was centrifuged under 12 000 r/min at 4 ℃ for 40 min, obtaining the precipitate which was washed twice with 0.02 mol/L phosphate buffer (goldlabeled antibody diluent) containing 0.5% BSA and 0.3% surfactant with a pH at 7.2, and resuspended with the goldlabeled antibody diluent in a volume 1/10 of the initial colloidal gold volume. The obtained liquid was placed in an environment at 4 ℃ for later use.

(4) Lyophilization of colloidal goldlabeled monoclonal antibody

20 μl of colloidal goldlabeled monoclonal antibody against tetracyclines, 20 μl of colloidal goldlabeled monoclonal antibody against fluoroquinolones and 20 μl of colloidal goldlabeled monoclonal antibody against sulfonamides were added into microwell strips, respectively. The microwell strips were then added into a freeze drier, prefreezed at -50 ℃ for 3 h and vacuumdried for 15 h. They were finally taken out as the microwell agents carrying colloidal goldlabeled monoclonal antibody against tetracyclines, colloidal goldlabeled monoclonal antibody against fluoroquinolones and colloidal goldlabeled monoclonal antibody against sulfonamides, which were sealed and preserved.

(5) Treatment and assembly of various parts of the test strip

① Treatment of sample absorbent pads.

Sample absorbent pads were soaked in a 0.2 mol/L phosphate buffer containing calf serum 2% and surfactant 1% with a pH at 7.2 for 2 h, and dried at 37 ℃ for 2 h.

② Treatment and coating of nitrocellulose membrane (NC membrance).

Artificial antigen of tetracyclines, fluoroquinolones and sulfonamides were diluted with 0.01 mol/L phosphate buffer with a pH at 7.2 to 1 mg/ml, and coated on detection line (T1), detection line (T2) and detection line (T3) of nitrocellulose membrane at a rate of 1.0 μl/cm, respectively. Goat antimouse immunoglobulin IgG was diluted with 0.01 mol/L phosphate buffer with a pH at 7.2 to 200 μg/ml, and coated on the quality control line (C) of the NC membrane at a rate of 1.0 μl/cm. The coated NC membrane was dried at 37 ℃for 2 h.

③ Assembly of the test strip.

After the sample absorbent pad, NC membrane, absorbent pad and PVC substrate were assembled, it was cut into 4 mm wide and used with the microwell agents lyophilized with colloidal goldlabeled monoclonal antibodies.

(6) Detection methods

200 μl of the milk sample solution was pipetted to microwells, and mixed uniformly with corresponding microwell agent. The microwells were heated in a mini metal bath at 45 ℃. One labeled test strip was inserted to one microwell at 45 ℃ with the handheld end upwards and the sample absorbent pad end downwards to completely immerse the sample absorbent pad into the sample. Timing was started when the liquid flew. After 8 min of reaction at 45 ℃, the liquid would vertically sequentially pass through the detection line and quality control line of the test strip upwards. The results could be judged according to the appearance and disappearance of red band and the shade of the color. The detection results are shown in Fig. 1.



Fig. 1Schematic diagram of detection results

(7) Performance verification of the test strip

① Test of sample detection limits.

Blank milk samples were added with tetracycline, fluoroquinolone and sulfonamide standards to the mass concentrations shown in Table 2, and a negative control was also set. The liquids were detected with test strips, and the sample detection limits were judged based on the test results.

② Test of false positive rate and false negative rate.

50 blank milk samples were detected with the test strip to calculate the false positive rate. 50 positive milk samples added with tetracycline, fluoroquinolone and sulfonamide standards to the detection limits were detected with the test strip to calculate the false negative rate.

③Test of specificity.

Other drugs commonly found in milk (melamine, chloramphenicol, erythromycin, streptomycin, neomycin, gentamicin, kanamycin, tylosin and tilmicosin) were diluted with 0.02 mol/L phosphate buffer with a pH at 7.2, and detected with the test strip. The specificity of the test strip was determined based on the detection results.

④Test of stability.

Enough test strips were preserved at 2-8 ℃, and a certain amount of test strips were taken out every other month for the determination of 10 blank milk samples, 10 positive milk samples added with tetracycline to 3 μg/L, 10 positive milk samples added with enrofloxacin to 0.2 μg/L and 10 positive milk samples added with sulfadiazine to 3 μg/L, with two repetitions. The stability of the test strip was judged according to the detection results.

Results and Analysis

Analytical analysis of colloidal gold quality

With doubledistilled water as a control, absorption curves and absorption peaks were measured by an ultravioletvisible spectrophotometer at 400-600 nm. The colloidal gold result was λmax=523 nm, as shown in Fig. 2. The transmission electron microscope observation showed that the prepared colloidal gold had a uniform particle size, and the 100 particles randomly measured showed a colloidal gold particle size of (20 ± 0.5) nm, as shown in Fig. 3.

Determination of the optimum antibody dosage for stabilizing colloidal gold

Taking the monoclonal antibody against tetracyclines as an example, the optimal antibody dosage for stabilizing colloidal gold is shown in Fig. 4. The no antibody tube (tube 1) and tubes 2-4 added with antibody not enough for stabilizing colloidal gold showed the coagulation phenomenon of turning from red to blue; the color of tube 9 without sodium chloride did not change; tubes 5-8 added with antibody enough or exceeding the amount for stabilizing colloidal gold maintained red constantly, and the antibody contained in tube 5 added with the least antibody was the minimum antibody dosage for stabilizing 1 ml of colloidal gold; and on this basis, the amount increased by 20% was the actual dosage of tobelabeled antibody, i.e., to stabilize 1 ml of colloidal gold liquid, 24 μg of monoclonal antibody against tetracyclines was added. The most suitable amounts of other two kinds of antibodies were: 24 μg (monoclonal antibody against fluoroquinolones) and 30 μg (monoclonal antibody against sulfonamides), respectively.



Fig. 2Scanning chart of colloidal gold by ultravioletvisible spectrophotometer

Yuping WAN et al. Study on Rapid Detection of Tetracyclines, Fluoroquinolones and Sulfonamides in Milk



Fig. 3Electron microscope scanning result of colloidal gold

Verification results of test strip performance

(1) Test of sample detection limits

According to the results in Table 2, the detection limits of the test strip for following drugs were: tetracycline, chlortetracycline and oxytetracycline 3 μg/L, doxycycline 2 μg/L, danofloxacin, ofloxacin and lomefloxacin 0.45 μg/L, pefloxacin, oxolinic acid and enrofloxacin 0.2 μg/L, flumequine 0.35 μg/L, norfloxacin 0.25 μg/L, enoxacin 0.4 Gg/L, ciprofloxacin 0.28 μg/L, sarafloxacin 1.0 μg/L, difloxacin 0.7 μg/L, marbofloxacin 1.2 μg/L, sulfadiazine 3 μg/L, sulfamethazine and sulfaclozine 4 μg/L, sulfametoxydiazine and sulfamonomethoxine 2 μg/L, sulfamethazine and sulfachlorpyridazine 14 μg/L, sulfadimethoxypyrimidine 8 μg/L, sulfamethythiadiazole and sulfaquinoxaline 7 μg/L, sulfamethoxazole and sulfathiazole 40 μg/L, sulfamethoxypyridazine 70 μg/L.

Table 2Measurement results of detection limits

Added drug Massconcentrationsμg/LDetection result

Tetracycline, chlortetracy0T1：(－) T2：(－) T3：(－)

cline, oxytetracycline1.5T1：(－) T2：(－) T3：(－)

3T1：(＋) T2：(－) T3：(－)

6T1：(＋) T2：(－) T3：(－)

Doxycycline0T1：(－) T2：(－) T3：(－)

1T1：(－) T2：(－) T3：(－)

2T1：(＋) T2：(－) T3：(－)

4T1：(＋) T2：(－) T3：(－)

Danofloxacin, ofloxacin, 0T1：(－) T2：(－) T3：(－)

lomefloxacin0.225T1：(－) T2：(－) T3：(－)

0.45T1：(－) T2：(＋) T3：(－)

0.9T1：(－) T2：(＋) T3：(－)

Pefloxacin, oxolinic acid, 0T1：(－) T2：(－) T3：(－)

enrofloxacin0.1T1：(－) T2：(－) T3：(－)

0.2T1：(－) T2：(＋) T3：(－)

0.4T1：(－) T2：(＋) T3：(－)

Flumequine 0T1：(－) T2：(－) T3：(－)

0.175T1：(－) T2：(－) T3：(－)

0.35T1：(－) T2：(＋) T3：(－)

0.7T1：(－) T2：(＋) T3：(－)

Norfloxacin 0T1：(－) T2：(－) T3：(－)

0.125T1：(－) T2：(－) T3：(－)

0.25T1：(－) T2：(＋) T3：(－)

0.5T1：(－) T2：(＋) T3：(－)

Enoxacin0T1：(－) T2：(－) T3：(－)

0.2T1：(－) T2：(－) T3：(－)

0.4T1：(－) T2：(＋) T3：(－)

0.8T1：(－) T2：(＋) T3：(－)

Ciprofloxacin0T1：(－) T2：(－) T3：(－)

0.14T1：(－) T2：(－) T3：(－)

0.28T1：(－) T2：(＋) T3：(－)

0.56T1：(－) T2：(＋) T3：(－)

Sarafloxacin0T1：(－) T2：(－) T3：(－)

0.5T1：(－) T2：(－) T3：(－)

(Continued)

(Table 2)

Added drug Massconcentrationsμg/LDetection result

1.0T1：(－) T2：(＋) T3：(－)

2.0T1：(－) T2：(＋) T3：(－)

Difloxacin0T1：(－) T2：(－) T3：(－)

0.35T1：(－) T2：(－) T3：(－)

0.7T1：(－) T2：(＋) T3：(－)

1.4T1：(－) T2：(＋) T3：(－)

Marbofloxacin0T1：(－) T2：(－) T3：(－)

0.6T1：(－) T2：(－) T3：(－)

1.2T1：(－) T2：(＋) T3：(－)

2.4T1：(－) T2：(＋) T3：(－)

Sulfadiazine 0T1：(－) T2：(－) T3：(－)

1.5T1：(－) T2：(－) T3：(－)

3T1：(－) T2：(－) T3：(＋)

6T1：(－) T2：(－) T3：(＋)

Sulfamerazine, sulfaclozine0T1：(－) T2：(－) T3：(－)

2T1：(－) T2：(－) T3：(－)

4T1：(－) T2：(－) T3：(＋)

8T1：(－) T2：(－) T3：(＋)

Sulfametoxydiazine, 0T1：(－) T2：(－) T3：(－)

sulfamonomethoxine1T1：(－) T2：(－) T3：(－)

2T1：(－) T2：(－) T3：(＋)

4T1：(－) T2：(－) T3：(＋)

Sulfamethazine, 0T1：(－) T2：(－) T3：(－)

sulfachlorpyridazine7T1：(－) T2：(－) T3：(－)

14T1：(－) T2：(－) T3：(＋)

28T1：(－) T2：(－) T3：(＋)

Sulfadimethoxypyrimidine0T1：(－) T2：(－) T3：(－)

4T1：(－) T2：(－) T3：(－)

8T1：(－) T2：(－) T3：(＋)

16T1：(－) T2：(－) T3：(＋)

Sulfamethythiadiazole, 0T1：(－) T2：(－) T3：(－)

sulfaquinoxaline3.5T1：(－) T2：(－) T3：(－)

7T1：(－) T2：(－) T3：(＋)

14T1：(－) T2：(－) T3：(＋)

Sulfamethoxazole, 0T1：(－) T2：(－) T3：(－)

sulfathiazole20T1：(－) T2：(－) T3：(－)

40T1：(－) T2：(－) T3：(＋)

80T1：(－) T2：(－) T3：(＋)

Sulfamethoxypyridazine 0T1：(－) T2：(－) T3：(－)

35T1：(－) T2：(－) T3：(－)

70T1：(－) T2：(－) T3：(＋)

140T1：(－) T2：(－) T3：(＋)

(2) Test of false positive rate and false negative rate

The test results of 50 blank milk samples showed that two samples were positive, the rest were all negative, and the false positive rate was less than 5%; and the test results of 50 positive milk samples showed that all the samples were positive, and the false negative rate was 0.

(3) Test of specificity

The results showed that for melamine, chloramphenicol, erythromycin, streptomycin, neomycin, gentamicin, kanamycin, tylosin and tilmicosin at a concentration of 500 μg/L, the test results of the test strip were still negative, suggesting that the test strip does not crossreact with these drugs, and the specificity is better.



Fig. 4Test results of proportions of colloidal gold and monoclonal antibody against tetracyclines

(4) Test of stability

The results showed that the test results of the blank milk samples were negative from the production date to the 12th month, and the positive milk samples were all positive; and after 12 months, the test strips showed false positive and failure. It can be seen that the test strip has good stability and can be stored for at least 12 months at 2-8 ℃.

Conclusions

The colloidal gold test strip prepared in this study can simultaneously detect 4 tetracyclines, 13 fluoroquinolones and 13 sulfonamides in milk. The detection limits all meet the national residue limit requirements; and it has the false positive rate≤5% and the false negative rate=0, no crossreaction with other drugs commonly found in milk, and short detection time, and thus can be used as a main product for screening tetracycline, fluoroquinolone and sulfonamide residues in dairy products by dairy farms, dairy companies, government monitoring departments and large food circulation areas, so the antibiotic problem in milk can be effectively monitored.

At the same time, in this study, the colloidal goldlabeled antibody was directly lyophilized in the microwell agent, and during the detection process, the goldlabeled antibody was sufficiently contacted with the sample liquid to be tested, and fully reacted, thereby reducing errors, increasing the reaction sensitivity of the entire system and facilitating the analysis of drug residues in samples. It also abandons the plastic card case of the traditional test strip and installs in the agent bucket only in the form of "bare strip", thereby saving space and reducing production and transportation cost. Although the improvement of the structure improves the sensitivity of the test strip, for the positive sample, it is necessary to confirm by an instrument method such as LCMS/MS as the result of the color judgment is subjective and false positive phenomenon may occur.

References

［1］ HUANG C, WU QM, SUN ZY, et al. Research progress on rapid detection of antibiotic residues in milk［J］. Food Research and Development, 2016, 37(23): 221-224. (in Chinese)

［2］ JIANG L, ZENG K, SHAO J, et al. Advances in rapid analysis methods for tetracycline antibiotics［J］. Jiangsu Agricultural Sciences, 2018, 46(8): 15-21. (in Chinese)

［3］ BLONDEAU JM. Fluoroquinolones: mechanism of action, classification, and development of resistance［J］. Survey of Ophthalmology, 2004, 49(2): S73-S78.

［4］ YU M. Harm and current status of sulfonamide residues in aquaculture［J］. Technical Advisor for Animal Husbandry, 2015(2): 133-133. (in Chinese)

［5］ Veterinary Bureau of the Ministry of Agriculture. Notice of the Ministry of Agriculture on the Monitoring Program for Veterinary Drug Residues in Animals and Animal Products in 2018［EB/OL］. http://www.moa.gov.cn/gk/ghjh_1/201801/t20180129_6135855.htm, 2018-01-22. (in Chinese)

［6］ BARHAM R, BLACK WD, CLAXTON J, et al. Rapid assay for detecting sulfonanides in tissues of slaughtered animals［J］. Food protection, 2001, 64(10): 1565-1573.

［7］ HUANG XR, ZHENG J, LI SS, et al. A rapid and simple microbiology method of determination of the quinolones residue in eel samples with a sensitive bacteria［J］. Freshwater Fisheries, 2005, 35(4): 3-6. (in Chinese)

［8］ XU H, MI HY, GUAN MM, et al. Residue analysis of tetracyclines in milk by HPLC coupled with hollow fiber membranesbased dynamic liquidliquid microextraction［J］. Food Chemistry, 2017, 232: 198-202.

［9］ JIANG XH, YANG SY, SHEN RM. Determination of 4 kinds of fluoroquinolones in egg by high performance liquid chromatography［J］. Food Safety and Quality Detection Technology, 2016, 7(12): 4978-4981. (in Chinese)

［10］ KAN GL, WANG XJ, WEI HT, et al. Simultaneous determination of 15 kinds of quinolone residues and17 kinds of sulfonamide residues in honey by liquid chromatographytandem mass spectrometry［J］. Food Safety and Quality Detection Technology, 2017, 8(9): 3571-3578. (in Chinese)

［11］ GUIDI LR, SANTOS FA, RIBEIRO A, et al. Quinolones and tetracyclines in aquaculture fish by a simple and rapid LCMS/MS method［J］. Food Chemistry, 2018, 245: 1232-1238.

［12］ WANG N. HPCE for the determination of anthraquinones and sulfonamides, fluoroquinolones residues［D］. Baoding: Hebei University, 2015. (in Chinese)

［13］ SONG YZ, DONG Q, XING GX, et al. Application of immunoassay in the sulfonamides residues detection［J］. Journal of Henan Agricultural Sciences, 2017, 46(10): 8-12. (in Chinese)

［14］ TUFA RA, PINACHO DG, PASCUAL N, et al. Development and validation of an enzyme linked immunosorbent assay for fluoroquinolones in animal feeds［J］. Food Control, 2015, 57: 195-201.

［15］ LI N, ZHANG YN, LIN T, et al. Analysis of sulfonamide, quinolones, steroid hormones and tetracyclines residues in milk by ultra performance liquid chromatographymass spectrometry［J］. Journal of Instrumental Analysis, 2016, 35(6): 714-718. (in Chinese)

［16］ WANG S, ZHANG J, SHAO B. Analysis of chloramphenicol, sulfonamides, fluoroquinolones, tetracyclines and macrolides in sewage sludge by ultra performance liquid chromatography tandem mass spectrometry［C］//2014 Annual Academic Conference of Chinese Society For Environmental Sciences, 2014. (in Chinese)

［17］ GBYLIKSIKORSKA M, POSYNIAK A, SNIEGOCKI T, et al. Liquid chromatography–tandem mass spectrometry multiclass method for the determination of antibiotics residues in water samples from water supply systems in foodproducing animal farms［J］. Chemosphere, 2015, 119(11): 8-15.

［18］ HU FY, HE LM, YANG JW, et al. Determination of 26 veterinary antibiotics residues in water matrices by lyophilization in combination with LCMS/MS［J］. Journal of Chromatography B, 2014, 949-950(4): 79-86.

［19］ SUN X, ZHAO X, JIAN T, et al. Preparation of goldlabeled antibody probe and its use in immunochromatography assay for detection of aflatoxin B1［J］. International Journal of Food Microbiology, 2005, 99(2): 185-194.