Development of Immunochromatographic Assay for the Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae Antibodies

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the cause of contagious caprine pleuropneumonia (CCPP), which is a highly significant respiratory disease in goats leading to significant economic losses in Africa and Asia. Currently available procedures for the diagnosis of CCPP have some limitations in sensitivity, specificity, operation time, requirement of sophisticated equipment or skilled personnel, and cost. In this study, we developed a rapid, sensitive, and specific colloidal gold-based immunochromatographic assay (GICA) strip for the efficient on-site detection of antibodies against Mccp in the serum within 10 min. For the preparation of this colloidal GICA strip, recombinant P20 protein, the membrane protein of Mccp, was expressed by Escherichia coli prokaryotic expression system after purification was used as the binding antigen in the test. The rabbit anti-goat immunoglobulin G labeled with the colloidal gold was used as the detection probe, whereas the goat anti-rabbit immunoglobulin G was coated on the nitrocellulose membrane as the control line. The concentration of the coating antibody was optimized, and the effectiveness of this colloidal GICA strip was evaluated. Our results proved that the detection limit of the test strip was up to 1:64 dilutions for the Mccp antibody-positive serum samples with no cross-reactivity with other pathogens commonly infecting small ruminants,including goat pox virus, peste des petits ruminants virus, foot-and-mouth disease virus type A, or other mycoplasmas. Moreover, the colloidal GICA strip was more sensitive and specific than the indirect hemagglutination assay for the detection of Mccp antibodies. The 106 clinical serum samples were detected by the colloidal GICA strip compared with the complement fixation test, demonstrating an 87.74% concordance with the complement fixation test. This novel colloidal GICA strip would be an effective tool for the cost-effective and rapid diagnosis of CCPP in the field.

Novel Latex Microsphere Immunochromatographic Assay for Rapid Detection of Cadmium Ion in Asparagus

Recently, concerns about heavy metal cadmium ion (Cd2+) residue in asparagus have been frequently reported, and there is an urgent need to develop an effective, sensitive, and rapid detection method for Cd2+. In this study, we innovatively combined molecular microbiology to carry out the comparative screening of Cd2+ chelators in a green, efficient, and specific way. The knock-out putative copper-transporter gene (pca1Δ) yeast strain with high sensitivity to Cd2+ was first used to screen the Cd2+ chelator, and the optimum chelator 1-(4-Isothiocyanatobenzyl)ethylenediamine-N,N,N,N′-tetraacetic acid (ITCBE) was obtained. Additionally, a rapid latex microsphere immunochromatographic assay (LMIA) was developed, based on the obtained monoclonal antibody (mAb) with high specificity and high affinity (affinity constant Ka = 1.83 × 1010 L/mol), to detect Cd2+ in asparagus. The 50% inhibitive concentration (IC50) of test strip was measured to be 0.2 ng/mL, and the limit of detection (IC10) for qualitative (LOD, for visual observation) and quantitative detection (LOQ, for data simulation) of the test strip was 2 ng/mL and 0.054 ng/mL, respectively. In all, the developed mAb-based LMIA shows a great potential for monitoring Cd2+ in asparagus, even in vegetable samples.

Rapid characterization of feline leukemia virus infective stages by a novel nested recombinase polymerase amplification (RPA) and reverse transcriptase-RPA

Feline leukemia virus (FeLV) is a major viral disease in cats, causing leukemia and lymphoma. The molecular detection of FeLV RNA and the DNA provirus are important for staging of the disease. However, the rapid immunochromatographic assay commonly used for antigen detection can only detect viremia at the progressive stage. In this study, nested recombinase polymerase amplification (nRPA) was developed for exogenous FeLV DNA provirus detection, and reverse transcriptase polymerase amplification (RT-RPA) was developed for the detection of FeLV RNA. The approaches were validated using 108 cats with clinicopathologic abnormalities due to FeLV infection, and from 14 healthy cats in a vaccination plan. The nRPA and RT-RPA assays could rapidly amplify the FeLV template, and produced high sensitivity and specificity. The FeLV detection rate in regression cats by nRPA was increased up to 45.8% compared to the rapid immunochromatographic assay. Hence, FeLV diagnosis using nRPA and RT-RPA are rapid and easily established in low resource settings, benefiting FeLV prognosis, prevention, and control of both horizontal and vertical transmission.