Lateral flow immunoassay strips based on europium(III) chelate microparticle for the rapid and sensitive detection of Trichinella spirali s infection in whole blood samples of pigs

Abstract

Trichinellosis is a major food-borne parasitosis caused by ingesting raw or semi-raw meat products from pigs infected with Trichinella spiralis (T. spiralis). Although China is the largest consumer of pork in the world, the current diagnostic method of T. spiralis is exclusively performed in a laboratory setting, due to its complexity and laborious procedure. Here, in order to solve the detection problems in the pig breeding industry, a rapid, sensitive, and on-site serological diagnosis method was developed. The novel lateral flow immunoassay strip (ICS) is based on europium(III) chelate microparticle (ECM) to detect T. spiralis-specific IgG antibody in the serum and whole blood samples from pigs. The structure of the blood-filtering pad and the conjugate pad was added to the ICS, allowing for whole blood samples to be detected and enabling on-site deployment. By comparing the detection results of the serum samples and the whole blood samples, the detection limit of this method was evaluated. Thereafter, this method was used to investigate Trichinella infection in Chongqing, Sichuan, Inner Mongolia, Guangxi, and Liaoning provinces of China, and the results were almost consistent with the standard method of artificial digestion. Taking advantage of its user-friendly procedure, short detection time (3 min), and sensitivity, the ECM-ICS could be employed for monitoring the epidemic of Trichinella infection and ensuring meat safety.

Keywords: Trichinella spiralis; europium(III) chelate microparticle; excretory–secretory; immunochromatographic strip; on-site detecting.

Figure 1

The schematic of europium(III) chelate microparticle-immunoassay strip (ECM-ICS). (A) Detecting the whole blood sample by ECM-ICS. (B) Schematic depiction of the construction of the ECM-ICS. (C) Immunochromatographic process of the ECM-ICS.

Figure 2

Optimization of the ECM-ICS. (A) Fluorescent particles sprayed on conjugate pads 1–3: 0.4, 0.8, and 1.2 µg. (B) The rate of ES antigens conjugated with fluorescent particles 1–4: 1:10, 1:20, 1:30, and 1:40. (C) The materials of conjugate pads 1–6: VL78, VL98, SB06, SB08, RB45, and RB65. (D) The materials of NC membranes 1–3: CN140, Millipore 135, and Millipore 90. ECM-ICS was analyzed by the TRF reader (A1, B1, C1, D1). Visual results of the ICS under ultraviolet light (A2, B2, C2, D2).

Figure 3

The dilution limit of whole blood samples by ECM-ICS. (A) ECM-ICS was analyzed by the TRF reader. (B) Visual results of ICS under ultraviolet light. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 represent the whole blood samples diluted 50, 100, 200, 400, 800, 1,600, 3,200, 6,400, 12,800, and 25,600 times.

Figure 4

Serum samples detected by ECM-ICS. (A) Samples from pigs infected with 400 Trichinella. (B) Samples from pigs infected with 600 Trichinella. (C) Samples from pigs infected with 600 Trichinella. (D) Samples from pigs infected with 800 Trichinella. (A1–5) 30, 35, 45, 60, and 90 dpi; (B1–6) 25, 30, 35, 45, 60, and 90 dpi; (C1–6) 25, 30, 35, 45, 60, and 90 dpi; (D1–7) 21, 25, 30, 35, 45, 60, and 90 dpi.

Figure 5

Comparison of ECM-ICS for detecting whole blood and serum samples. (A) Samples from pigs infected with Trichinella at 21 dpi. (B) Samples from pigs infected with Trichinella at 25 dpi. (C) Samples from pigs infected with Trichinella at 30 dpi. (D) Samples from pigs infected with Trichinella at 35 dpi. (A1, B1, C1, D1) ECM-ICS for detecting whole blood samples analyzed by the TRF reader; (A2, B2, C2, D2) ECM-ICS for detecting serum samples analyzed by the TRF reader;(A3, B3, C3, D3) the whole blood samples detected by ECM-ICS and the visual results under ultraviolet light.