Renli Zhang

Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes

Clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is a fish-borne zoonosis endemic in a number of countries. This article describes the development of a TaqMan based real-time PCR assay for detection of C. sinensis DNA in human feces and in fishes. Primers targeting the first internal transcribed spacer (ITS-1) sequence of the fluke were highly specific for C. sinensis, as evidenced by the negative amplification of closely related trematodes in the test with the exception of Opisthorchis viverrini. The detection limit of the assay was 1pg of purified genomic DNA, 5EPG (eggs per gram feces) or one metacercaria per gram fish filet. The assay was evaluated by testing 22 human fecal samples and 37 fish tissues microscopically determined beforehand, and the PCR results were highly in agreement with the microscopic results. This real-time PCR assay provides a useful tool for the sensitive detection of C. sinensis DNA in human stool and aquatic samples in China and other endemic countries where O. viverrini and Opisthorchis felineus are absent.

Selective molecularly imprinted stationary phases for Bisphenol A analysis prepared by modified precipitation polymerization

Bisphenol A (BPA)-imprinted polymeric microspheres were synthesized by modified precipitation polymerization (MPP) method. Influences of cross-linker, monomer, porogen volume, and agitation on polymerization were investigated. Proper amount of cross-linker ethyleneglycol-dimethacrylate (EGDMA) was critical to achieve narrowly dispersed microspheres. For template BPA, monomer 4-vinylpyridine (4-VP) was better than MAA to get the best imprinted effects. The optimum template/monomer ratio was 1:6. Increasing porogen volume increased size dispersity and decreased binding characters. Agitation increased coagulation and resulted in irregular particles. Microspheres with the best binding characters were used as selective stationary phase of chromatographic column to detect BPA in milk, pig urine, and chicken meat. Under optimal chromatographic conditions, the calibration graph was linear with R2 = 0.9994 in the range of 3-50 micromol/L. The LOD and LOQ were 1 and 3 micromol/L, respectively. When large amounts (20 mL or 20 g) of samples were analyzed, the recoveries ranged from 70.2 to 87.3% with RSD less than 4.85% in all samples spiked with 0.05-0.2 micromol/L BPA. The intra-day and inter-day RSD were less than 1.83 and 3.96%, respectively. Microspheres prepared by MPP are successfully used in molecularly imprinted polymer (MIP)-based analytical column to detect trace BPA in different biologic samples with acceptable accuracy and repeatability.

Angiostrongylus cantonensis: identification and characterization of microRNAs in male and female adults.

Angiostrongylus cantonensis causes eosinophilic meningitis and eosinophilic pleocytosis in humans and is of significant socio-economic importance globally. microRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in gene expression regulation, cellular function and defense, homeostasis and pathogenesis. They have been identified in a diverse range of organisms. The objective of this study was to determine and characterize miRNAs of female and male adults of A. cantonensis by Solexa deep sequencing. A total of 8,861,260 and 10,957,957 high quality reads with 20 and 23 conserved miRNAs were obtained in females and males, respectively. No new miRNA sequence was found. Nucleotide bias analysis showed that uracil was the prominent nucleotide, particularly at positions of 1, 10, 14, 17 and 22, approximately at the beginning, middle and the end of the conserved miRNAs. To our knowledge, this is the first report of miRNA profiles in A. cantonensis, which may represent a new platform for studying regulation of genes and their networks in A. cantonensis.

Monoclonal antibodies against excretory/secretory antigens of Angiostrongylus cantonensis.

In the present study, four murine monoclonal antibodies (MAbs) were generated against the excretory/secretory (ES) products of Angiostrongylus cantonensis adult worms; two represented IgG1 and two represented IgM MAbs, and they were designated 12D5, 15F8, 21B7 and 14G10, respectively. Immunoblotting revealed that all of the MAbs predominantly recognized a 98u2009kDa antigen in the ES products of A. cantonensis adult worms, and no cross reactions were found with the whole worm antigens of some other common parasites, namely, Schistosoma japonicum, Clonorchis sinensis, Paragonimus westermani, Ascaris lumbricoides, Trichinella spiralis, Anisakis sp., Echinococcus granulosus, Taenia solium, and Spirometra erinacei. Immunolocalization showed that all of the four MAbs reacted with the cuticle of the adult parasite, the external surface of its intestinal canal and reproductive organs, and its egg and first-stage larvae in the lungs of rats experimentally infected with A. cantonensis. The generation and characterization of four specific MAbs against A. cantonensis ES antigens provide foundation for the development of specific immunological diagnostic techniques for human infections with A. cantonensis.

Specific detection of Angiostrongylus cantonensis in the snail Achatina fulica using a loop-mediated isothermal amplification (LAMP) assay.

Angiostrongylus cantonensis, a rat lungworm, can cause eosinophilic meningitis and angiostrongyliasis in humans following ingestion of contaminated foods or intermediate/paratenic hosts with infective larvae. The snail Achatina fulica is one of the important intermediate hosts of A.xa0cantonensis and is commonly eaten by humans in some countries. In the present study, we developed a loop-mediated isothermal amplification (LAMP) method for the specific detection of A.xa0cantonensis in Ac. fulica. Primers for LAMP were designed based on the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA) of A.xa0cantonensis. Specificity tests showed that only the products of A.xa0cantonensis were detected when DNA samples of A.xa0cantonensis and the heterologous control samples Anisakis simplex s.s, Trichuris trichiura, Toxocara canis, Trichinella spiralis and Ascaris lumbricoides were amplified by LAMP. Sensitivity evaluation indicated that the LAMP assay is 10 times more sensitive than the conventional polymerase chain reaction (PCR) assay. The established LAMP assay is rapid, inexpensive and easy to be performed. It can be used in clinical applications for rapid and sensitive detection of A.xa0cantonensis in snails, which has implications for the effective control of angiostrongyliasis.

DEVELOPMENT OF A DOUBLE ANTIBODY SANDWICH ELISA ASSAY FOR THE DIAGNOSIS OF ANGIOSTRONGYLIASIS

abstract:u2003 With the use of 2 monoclonal antibodies (MAbs) against excretory/secretory (ES) antigens of adult Angiostrongylus cantonensis, a new method was developed for double antibody sandwich ELISA for the detection of circulating antigens (CAg). To evaluate the sensitivity of the new procedure, the CAg in sera of rats (80) and mice (15) infected with A. cantonensis, as well as CAg in sera of clinically confirmed angiostrongyliasis patients (70), were evaluated. Cross-reaction testing was used to determine the specificity of serum from patients infected with Ascaris lumbricoides, Trichinella spiralis, Toxoplasma gondii, Schistosoma japonicum, Paragonimus westermani, Clonorchis sinensis, Echinococcus granulosus, Spirometra, and Taenia solium, as well as normal healthy people. The results proved that the sensitivity and the specificity of the new method were totally effective for the detection of A. cantonensis CAg. The assay is highly sensitive, specific, and reproducible, with easy handling and excellent cost effectiveness, and thereby provides a new method for the accurate diagnosis of angiostrongyliasis.

Selenoprotein K modulate intracellular free Ca(2+) by regulating expression of calcium homoeostasis endoplasmic reticulum protein.

Selenoprotein K (SelK) is an 11-kDa selenoprotein, which may be involved in the regulation of oxidative stress, endoplasmic reticulum (ER) stress and immune response. To explore the function of SelK in the process of immune response, several short-hairpin RNAs (shRNA) were designed for the construction of recombinant plasmids to down-regulate the expression of SelK gene inxa0vitro. These shRNAs specifically and efficiently interfered with the expression of SelK at both mRNA and protein levels. The expression of calcium homoeostasis endoplasmic reticulum protein (CHERP) and the intracellular free Ca2+ concentration were significantly down-regulated in anti-CD3 stimulated SelK-knockdown cells. The expression of Interleukin 2 receptor alpha chain (IL-2Rα) and the secretion of Interleukin 4 (IL-4), which play a significant role in the process of T cell activation and proliferation, were also reduced in SelK-knockdown cells. Selenomethionine (Se-Met) at an optimum concentration of 5xa0μM could up-regulate SelK expression and reverse the change of the expression of CHERP and the intracellular free calcium caused by SelK-knockdown. These results hereby imply SelK may regulate the release of Ca2+ by CHERP and play an important role in the proliferation and differentiation of T cell by TCR stimulation.

Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection

Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis.

Seroprevalence of Angiostrongylus cantonensis Infection in Humans in China

Abstract A seroepidemiological survey was carried out in China during 2009–2010 to determine the extent of circulating antigens (CAg) for Angiostrongylus cantonensis in the Chinese population using the gold immunochromatographic assay, with the objective of elucidating the nationwide prevalence of angiostrongyliasis in China. A total of 1,730 blood samples was collected and assayed from the general adult population (the “general group”), and those involved in aquaculture or processing of snails Achatina fulica and Pomacea canaliculat (the “occupational group”) from 5 provinces (Fujian, Hunan, Guangdong, Guangxi, and Zhejiang) and 1 municipal city (Beijing). The overall seroprevalence for the “occupational group” was 7.4% (40/540), which was significantly higher (P < 0.001) than that of the “general group” (0.8%, 9/1,190). The seroprevalence in males (9.5%) was significantly higher than in females (4.2%) (P < 0.05). These results demonstrate that angiostrongyliasis represents a significant zoonotic disease in China, requiring the strengthening of food safety for control of this food-borne disease.

Highly Sensitive Naked-Eye Assay for Enterovirus 71 Detection Based on Catalytic Nanoparticle Aggregation and Immunomagnetic Amplification

Development of sensitive, convenient, and cost-effective virus detection product is of great significance to meet the growing demand of clinical diagnosis at the early stage of virus infection. Herein, a naked-eye readout of immunoassay by means of virion bridged catalase-mediated in situ reduction of gold ions and growth of nanoparticles, has been successfully proposed for rapid visual detection of Enterovirus 71 (EV71). Through tailoring the morphologies of the produced gold nanoparticles (GNPs) varying between dispersion and aggregation, a distinguishing color changing was ready for observation. This colorimetric detection assay, by further orchestrating the efficient magnetic enrichment and the high catalytic activity of enzyme, is managed to realize highly sensitive detection of EV71 virions with the limit of detection (LOD) down to 0.65 ng/mL. Our proposed method showed a much lower LOD value than the commercial ELISA for EV71 virion detection. Comparing to the current clinical gold standard polymerase chain reaction (PCR) method, our strategy provided the same diagnostic outcomes after testing real clinical samples. Besides, this strategy has no need of complicated sample pretreatment or expensive instruments. Our presented naked-eye immunoassay method holds a promising prospect for the early detection of virus-infectious disease especially in resource-constrained settings.