Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (22): 4578-4588.doi: 10.3864/j.issn.0578-1752.2024.22.015

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Establishment and Application of a Tube-Based Chemiluminescence Immunoassay Method for Detecting Antibodies Against Trichinella spiralis in Pigs

QIAN YanHong1(), SONG Shuai1(), WEN XiaoHui1, NIU RuiHui1, YANG YanQiu1, ZHENG BoBin1, YUAN ZiGuo2(), LUO ShengJun1()   

  1. 1 Institute of Animal Health, Guangdong Academy of Agricultural Sciences/Guangdong Province Key Laboratory of Livestock Diseases Prevention/Guangdong Scientific Observation and Experiment Station for Veterinary Drugs and Diagnostic Technologies, Ministry of Agriculture and Rural Areas, Guangzhou 510640
    2 College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642
  • Received:2024-05-28 Accepted:2024-07-23 Online:2024-11-16 Published:2024-11-22
  • Contact: YUAN ZiGuo, LUO ShengJun

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Abstract:

【Background】 Trichinella spiralis is a foodborne zoonotic parasitic nematode, listed as one of the seven major foodborne parasites in the world. In domestic animals, Trichinella spiralis mainly infects pigs and can be transmitted to humans through pork and by-products. It not only causes huge economic losses to China's pig farming industry, but also seriously endangers human health and public health safety.【Objective】This study aimed to establish a highly sensitive, specific, and rapid automated serological detection method for Trichinella spiralis in pigs.【Method】This study utilized gene cloning and prokaryotic expression techniques to construct a prokaryotic expression vector, obtained the recombinant protein of Trichinella spiralis 14-3-3 through induction of expression, and verified its reactivity. A chemiluminescence enzyme immunoassay method was established by encapsulating magnetic particles with prepared recombinant proteins as detection antigens, and the conditions for the established method were optimized. Finally, based on the optimized method, the critical values were defined and the specificity, sensitivity, and repeatability were determined; by testing 1 000 clinical serum samples of pigs from different pig farms in Guangdong and Guizhou provinces, the practicality and compliance rate of this testing method were evaluated.【Result】The recombinant protein Ts14-3-3 was successfully expressed and purified, which could specifically recognize antibodies in positive serum of Trichinella spiralis in pigs. The established detection method has been optimized, with the optimal pH value of the buffer solution being 5 and the optimal encapsulation amount of the Ts14-3-3 recombinant protein being 5 μg·mg-1-beads, the optimal dosage of the activator was 50 μg·mg-1-beads, and 1%BSA has the best sealing effect. The optimal dilution for enzyme-labeled antibodies was 1:40 000, the optimal reaction time was 10 minutes, the optimal incubation time for the sample to be tested was 5 minutes, and the optimal enzymatic reaction time was 2 minutes. Finally, after evaluation, the critical chemiluminescence value of this detection method was 374 185 RLU, and there was no cross-reaction with antibodies against Toxoplasma gondii, coccidia, Pseudorabies, and foot-and- mouth disease in pigs, as well as antibodies against porcine reproductive and respiratory syndrome, classical swine fever, pseudorabies, and foot-and-mouth disease. Compared with existing ELISA detection methods, this detection method had higher sensitivity, with intra - and inter-batch repeatability of less than 10%; the positive rate of trichinella antibodies in 1000 clinical pig serum samples detected was 0.6%, and a total compliance rate of 98.18% with commercially available ELISA kits. 【Conclusion】This study successfully established a tube-based chemiluminescence immunoassay method for detecting antibodies against Trichinella spiralis in pigs. The detection method had strong specificity, and good stability, and was more sensitive and rapid, and had a high clinical detection accuracy compared wtih ELISA methods.

Key words: Trichinella spiralis, serological diagnosis, Ts14-3-3, prokaryotic expression, tubular chemiluminescence immunoassay

Table 1

Primers sequence"

引物名称
Primer 		引物序列
Primers sequence (5'-3')		 限制性内切酶
Restrictive endonuclease
Ts14-3-3-F GGACAGCAAATGGGTCGCCGC GGATCCATGACCGAAAAGGAAGACATAGTT BamH I
Ts14-3-3-R GGTGGTGGTGGTGGTGCCG CTCGAGCTACTGCCCAGCGGCTGTAT Xho I

Fig. 1

Recombinant protein-induced expression M: 180 kDa pre-stained protein marker; 1: Uninduced PET-28 empty bacterial solution; 2: Induced pET-28a empty bacterial solution; 3: Uninduced pET-28a-Ts14-3-3 recombinant bacterial solution; 4: Induced pET-28a- Ts14-3-3 recombinant bacterial solution"

Fig. 2

Purification of recombinant protein M: 180 kDa pre-stained protein marker; 1: The supernatant of pET-28a- Ts14-3-3 recombinant bacterial cells induced by ultrasound; 2-4: Transflow fluid 1-3; 5-7: Washing solution 1-3; 8-10: Elution solution 1-3"

Fig. 3

Western blot identification of recombinant protein M: 180 kDa pre-stained protein marker; 1: Porcine Trichinella spiralis positive serum was used for primary antibody incubation; 2: Incubate with negative serum of Trichinella spiralis as primary antibody"

Table 2

Optimization results of magnetic particle (MPs) coupled antigen conditions"

结合缓冲液的pH
Combining the pH value of the buffer solution		 重组蛋白包被量
Recombinant protein coating amount		 EDC溶液和NHS溶液使用量
Usage of EDC solution and NHS solution		 封闭剂
Sealing agent
优化结果
Optimization result		 5 5 μg·mg-1-beads 50 μg·mg-1-beads 1%BSA

Fig. 4

Optimization of dilution and reaction time for enzyme-labeled antibodies A: Optimization of dilution of enzyme-labeled antibodies; B: Optimization of incubation time for test samples; C: Optimization of incubation time for enzyme-labeled antibodies; D: Optimization of chemiluminescence enzyme-catalyzed reaction time"

Fig. 5

ROC curve"

Fig. 6

The specificity test"

Table 3

The sensitivity test"

稀释度
Dilution degree		 CLIA ELISA
化学发光值
Chemiluminescence value (RLU)		 结果判定
Result determination		 OD450nm值
OD450nm value		 结果判定
Result determination
1:1 3 648 203 ＋ 2.242 ＋
1:2 3 579 619 ＋ 2.261 ＋
1:4 2 790 863 ＋ 1.161 ＋
1:8 2 355 127 ＋ 0.158 －
1:16 1 697 823 ＋ 0.089 －
1:32 1 107 413 ＋ 0.075 －
1:64 634 751 ＋ 0.052 －
1:128 286 312 － 0.048 －
1:256 77 946 － 0.047 －
1:512 74 670 － 0.044 －
1:1 024 48 498 － 0.047 －
1:2 048 35 766 － 0.045 －

Table 4

Results of intra-batch repeatability testing"

样本编号
Sample number		 化学发光值 Chemiluminescence value (RLU) 平均值
Average value		 标准差
Standard deviation		 变异系数
Coefficient of variation (%)
A B C
1 358 9182 3 446 347 3 657 215 3 564 248 107 622 3.02
2 1 823 223 1 748 002 1 779 230 1 783 485 37 790 2.12
3 2 619 698 2 651 526 2 516 291 2 595 838 70 704 2.72
4 137 198 150 790 151 496 146 494 8 059 5.50
5 158 929 179 714 156 394 165 012 12 794 7.75
6 150 155 168 542 167 274 161 990 10 269 6.34

Table 6

Clinical sample testing results"

临床血清样本Clinical serum sample 广东Guangdong 贵州Guizhou 总计Total
阳性Positive 0 6 6
阴性Negative 890 104 994
总计Total 890 110 1000
阳性率The positive rate (%) 0 5.45 0.6

Table 7

Compliance test"

检测方法
Detection method		 CLIA
阳性Positive 阴性Negative 总计Total
ELISA 阳性Positive 4 0 4
阴性Negative 2 104 106
总计Total 6 104 110
阳性符合率The positive conformity rate (%) 100.00
阴性符合率The negative conformity rate (%) 98.11
总符合率The conformity rate (%) 98.18
[1]
秦建华, 张龙现. 动物寄生虫病学. 北京: 中国农业大学出版社, 2013: 168-171.
QIN J H, ZHANG L X. Animal parasitology. Beijing: China Agricultural University Press, 2013: 168-171. (in Chinese)
[2]
KHURANA S, DATTA P, SHARMA B, SINGH C, MEWARA A, JOHNSON N, PILANIA R K, SINGH S, SEHGAL R. Clinical and laboratory profile of trichinellosis from a non-endemic country. Indian Journal of Medical Microbiology, 2021, 39(2): 235-239.
[3]
GLAZUNOV Y V, VINOGRADOVA Y A. Epidemiology study of Trichinella spiralis infection in tyumen region. Archives of Razi Institute, 2023, 78(2): 515-521.
[4]
BASSO W, MARREROS N, HOFMANN L, SALVISBERG C, LUNDSTRÖM-STADELMANN B, FREY C F. Evaluation of the PrioCHECK™ Trichinella AAD kit to detect Trichinella spiralis, T. britovi, and T. pseudospiralis larvae in pork using the automated digestion method Trichomatic-35. Parasitology International, 2022, 86: 102449.
[5]
ROSTAMI A, GAMBLE H R, DUPOUY-CAMET J, KHAZAN H, BRUSCHI F. Meat sources of infection for outbreaks of human trichinellosis. Food Microbiology, 2017, 64: 65-71.

doi: S0740-0020(16)30631-1 pmid: 28213036
[6]
MALONE C J, OKSANEN A, MUKARATIRWA S, SHARMA R, JENKINS E. From wildlife to humans: The global distribution of Trichinella species and genotypes in wildlife and wildlife-associated human trichinellosis. International Journal for Parasitology: Parasites and Wildlife, 2024, 24: 100934.
[7]
BUFFONI L, CANO-TERRIZA D, JIMÉNEZ-MARTÍN D, JIMÉNEZ- RUIZ S, MARTÍNEZ-MORENO Á, MARTÍNEZ-MORENO F J, ZAFRA R, PÉREZ-CABALLERO R, RISALDE M Á, GÓMEZ- GUILLAMÓN F, GARCÍA-BOCANEGRA I. Serosurveillance of Trichinella sp. in wild boar and Iberian domestic suids in Mediterranean ecosystems of southwestern Spain. Zoonoses and Public Health, 2024, 71(2): 191-199.
[8]
VUTOVA K, VELEV V, CHIPEVA R, YANCHEVA N, PETKOVA S, TOMOV T, POZIO E, ROBERTSON L J. Clinical and epidemiological descriptions from trichinellosis outbreaks in Bulgaria. Experimental Parasitology, 2020, 212: 107874.
[9]
MOHAMMED E S, YOUSEEF A G, MUBARAK A G, MAWAS A S, KHALIFA F A, FELEFEL W. Epidemiological perspective associated with principal risk factors of Trichinella spiralis infection in pigs and humans in Egypt. Veterinary World, 2022: 1430-1437.
[10]
CUPERLOVIC K, DJORDJEVIC M, PAVLOVIC S. Re-emergence of trichinellosis in southeastern Europe due to political and economic changes. Veterinary Parasitology, 2005, 132(1/2): 159-166.
[11]
VILLEGAS-PÉREZ J, NAVAS-GONZÁLEZ F J, SERRANO S, GARCÍA-VIEJO F, BUFFONI L. A quality assurance discrimination tool for the evaluation of satellite laboratory practice excellence in the context of the European official meat inspection for Trichinella spp. Foods, 2023, 12(22): 4186.
[12]
N’DA K M, GBATI O B, DAHOUROU L D, BEHOU N E S, TRAORE A, KUNGU J. Pigs' management practices and exposure to Trichinella spp. in pigs and warthogs in the northern area of Senegal. Veterinary World, 2022: 2253-2258.
[13]
LUPU M, PAVEL R, LAZUREANU V, POPOVICI E, OLARIU T. Trichinellosis in hospitalized patients from Western Romania: a retrospective study. Experimental and Therapeutic Medicine, 2021, 22(2): 895.
[14]
梁恒之. 胶体金免疫层析技术(GICA)在猪病检测中的应用研究进展. 浙江畜牧兽医, 2016, 41(6): 11-14.
LIANG H Z. Research progress on application of colloidal gold immunochromatography (GICA) in pig disease detection. Zhejiang Journal Animal Science and Veterinary Medicine, 2016, 41(6): 11-14. (in Chinese)
[15]
GÓMEZ-MORALES M Á, CHERCHI S, LUDOVISI A. Serological testing for Trichinella infection in animals and man: Current status and opportunities for advancements. Food and Waterborne Parasitology, 2022, 27: e00165.
[16]
孙宇, 张亮, 王子慰. 化学发光免疫分析方法研究进展. 中国高新区, 2017 (18):40.
SUN Y, ZHANG L, WANG Z W. Progress in chemiluminescence immunoassay. Science & Technology Industry Parks, 2017 (18):40. (in Chinese)
[17]
苑淑贤, 姚新华, 任科研, 李萌, 杨金生, 苑冬梅, 李煜洁, 常军亮, 杨淑苹. 猪旋毛虫病胶体金检测试纸条的制备. 中国生物制品学杂志, 2011, 24(8): 957-958, 963.
YUAN S X, YAO X H, REN K Y, LI M, YANG J S, YUAN D M, LI Y J, CHANG J L, YANG S P. Preparation of colloidal gold test strip for swine trichinellosis. Chinese Journal of Biologicals, 2011, 24(8): 957-958, 963. (in Chinese)
[18]
XU L, SUO X Y, ZHANG Q, LI X P, CHEN C, ZHANG X Y. ELISA and chemiluminescent enzyme immunoassay for sensitive and specific determination of lead (II) in water, food and feed samples. Foods, 2020, 9(3): 305.
[19]
程博文, 张宏顺. 化学发光免疫分析技术的应用进展. 中国工业医学杂志, 2022, 35(3): 237-240.
CHENG B W, ZHANG H S. Application progress of chemiluminescence immunoassay. Chinese Journal of Industrial Medicine, 2022, 35(3): 237-240. (in Chinese)
[20]
封莹洁, 张同锋, 罗娟, 尚佳静, 孟鸽, 于晓慧, 蒋文明, 刘华雷, 范春艳, 李阳. 化学发光免疫分析法简介及其在动物疫病检测中的研究进展. 中国动物检疫, 2023, 40(12): 73-79.
FENG Y J, ZHANG T F, LUO J, SHANG J J, MENG G, YU X H, JIANG W M, LIU H L, FAN C Y, LI Y. Introduction to chemiluminescence immunoassay and its application in detection of animal diseases. China Animal Health Inspection, 2023, 40(12): 73-79. (in Chinese)
[21]
包艳芳, 蒋韬, 何莉, 吕律, 李涛善, 李昕, 孙燕燕, 杨光, 魏婷, 潘晓乐, 林密. 口蹄疫病毒O型全自动磁微粒CLIA抗体定量检测方法的建立. 中国农业科学, 2024, 57(04):810-819. doi: 10.3864/j.issn.0578-1752.2024.04.014.
BAO Y F, JIANG T, HE L, LÜ L, LI T S, LI X, SUN Y Y, YANG G, WEI T, PAN X L, LIN M. Development of a full-automated magnetic particle chemiluminescence immunoassay assay for quantitative detection of antibodies against foot and mouth disease virus serotype O. Scientia Agricultura Sinica, 2024, 57 (04):810-819. doi: 10.3864/j.issn.0578-1752.2024.04.014. (in Chinese)
[22]
POZIO E. Trichinella spp. imported with live animals and meat. Veterinary Parasitology, 2015, 213(1/2): 46-55.
[23]
JI Y F, HE Z J, LI N J, LI C, XU T. Green production efficiency of China’s hog breeding industry: Spatial divergence and its driving factors. PLoS ONE, 2023, 18(11): e0288176.
[24]
LOBANOV V A, KONECSNI K A, PURVES R W, SCANDRETT W B. Performance of indirect enzyme-linked immunosorbent assay using Trichinella spiralis-derived Serpin as antigen for the detection of exposure to Trichinella spp. in swine. Veterinary Parasitology, 2022, 309: 109744.
[25]
MASOORI L, FALAK R, MOKHTARIAN K, BANDEHPOUR M, RAZMJOU E, JALALLOU N, JAFARIAN F, AKHLAGHI L, MEAMAR A R. Production of recombinant 14-3-3 protein and determination of its immunogenicity for application in serodiagnosis of strongyloidiasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 2019, 113(6): 326-331.

doi: 10.1093/trstmh/trz006 pmid: 30856271
[26]
YANG J, PAN W, SUN X M, ZHAO X, YUAN G, SUN Q, HUANG J J, ZHU X P. Immunoproteomic profile of Trichinella spiralis adult worm proteins recognized by early infection sera. Parasites & Vectors, 2015, 8(1): 20.
[27]
SUN T Q, ZHAO Z Q, LIU W T, XU Z H, HE H W, NING B A, JIANG Y Q, GAO Z X. Development of sandwich chemiluminescent immunoassay based on an anti-staphylococcal enterotoxin B Nanobody-Alkaline phosphatase fusion protein for detection of staphylococcal enterotoxin B. Analytica Chimica Acta, 2020, 1108: 28-36.

doi: S0003-2670(20)30068-4 pmid: 32222241
[28]
DENG J Q, YANG M Z, WU J, ZHANG W, JIANG X Y. A self-contained chemiluminescent lateral flow assay for point-of-care testing. Analytical Chemistry, 2018, 90(15): 9132-9137.

doi: 10.1021/acs.analchem.8b01543 pmid: 30004664
[29]
张建锋. 磁微粒-吖啶酯化学发光游离甲状腺素检测试剂的研制及应用. 国际检验医学杂志, 2016, 37(19): 2773-2775.
ZHANG J F. Development and application of magnetic particle- acridine ester chemiluminescence free thyroxine detection reagent. International Journal of Laboratory Medicine, 2016, 37(19): 2773-2775. (in Chinese)
[30]
WANG Z L, XIANYU Y L, ZHANG Z, GUO A L, LI X J, DONG Y Z, CHEN Y P. Background signal-free magnetic bioassay for food- borne pathogen and residue of veterinary drug via Mn(VII)/Mn(II) interconversion. ACS Sensors, 2019, 4(10): 2771-2777.
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