A recombinase polymerase amplification-SYBR Green I assay for the rapid and visual detection of Brucella.

Brucellosis is a zoonosis caused by Brucella, which poses a great threat to human health and animal husbandry. Pathogen surveillance is an important measure to prevent brucellosis, but the traditional method is time-consuming and not suitable for field applications. In this study, a recombinase polymerase amplification-SYBR Green I (RPAS) assay was developed for the rapid and visualized detection of Brucella in the field by targeting BCSP31 gene, a conserved marker. The method was highly specific without any cross-reactivity with other common bacteria and its detection limit was 2.14 × 104 CFU/mL or g of Brucella at 40 °C for 20 min. It obviates the need for costly instrumentation and exhibits robustness towards background interference in serum, meat, and milk samples. In summary, the RPAS assay is a rapid, visually intuitive, and user-friendly detection that is highly suitable for use in resource-limited settings. Its simplicity and ease of use enable swift on-site detection of Brucella, thereby facilitating timely implementation of preventive measures.

A rapid and sensitive triplex-recombinase polymerase amplification for simultaneous differentiation of Brucella abortus, Brucella melitensi s, and Brucella suis in sera and foods.

Brucella is the causative agent of brucellosis and can be transmitted to humans through aerosolized particles or contaminated food. Brucella abortus (B. abortus), Brucella melitensis (B. melitensis), and Brucella suis (B. suis) are the most virulent of the brucellae, but the traditional detection methods to distinguish them are time-consuming and require high instrumentation. To obtain epidemiological information on Brucella during livestock slaughter and food contamination, we developed a rapid and sensitive triplex recombinant polymerase amplification (triplex-RPA) assay that can simultaneously detect and differentiate between B. abortus, B. melitensis, and B. suis. Three pairs of primers (B1O7F/B1O7R, B192F/B192R, and B285F/B285R) were designed and screened for the establishment of the triplex-RPA assay. After optimization, the assay can be completed within 20 min at 39°C with good specificity and no cross-reactivity with five common pathogens. The triplex-RPA assay has a DNA sensitivity of 1-10 pg and a minimum detection limit of 2.14 × 104-2.14 × 105 CFU g-1 in B. suis spiked samples. It is a potential tool for the detection of Brucella and can effectively differentiate between B. abortus, B. melitensis, and B. suis S2, making it a useful tool for epidemiological investigations.

Tyrosol inhibits NF-κB pathway in the treatment of enterotoxigenic Escherichia coli-induced diarrhea in mice.

Tyrosol is one of the main polyphenol compounds in white wine and extra virgin olive oil (EVOO), which plays an antioxidant and anti-inflammatory role in vitro. In the present study, we investigated the possible anti-inflammatory mechanism of tyrosol in Escherichia coli (ETEC)-induced diarrhea in mice. ICR mice were randomly divided into control group, ETEC group, and ETEC + Tyrosol group with 10 mice in each group. In addition to the control group, a bacterial diarrhea model was induced in mice by continuous administration of 0.2 ml × 109 CFU/ml ETEC. After 7 days, the ETEC + Tyrosol group was given tyrosol (20 mg/kg) once a day by gavage, during which the body weight of mice and the degree of diarrhea were measured daily. On the 15th day, all animals in this experiment were sacrificed, colon tissue was collected, and colon length was recorded. Our results indicate that tyrosol significantly attenuated the extent of ETEC-induced diarrhea, including inhibition of pro-inflammatory cytokine, repair of the intestinal epithelial mechanical barrier, and significant inhibition of NF-κB activation. This finding is helpful for the development and further application of tyrosol in the treatment of diarrhea.

A comparison study of superovulation strategies for C57BL/6J and B6D2F1 mice in CRISPR-Cas9 mediated genome editing.

Reproductive techniques such as superovulation and in vitro fertilisation (IVF) have been widely used in generating genetically modified animals. The current gold standard for superovulation in mice is using coherent treatments of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). An alternative method using inhibin antiserum (IAS) instead of eCG has been recently reported. Here, we evaluate different superovulation strategies in C57BL/6J and B6D2F1 mice. Firstly, we found that using 5-week-old C57BL/6J and 4-week-old B6D2F1 donors could achieve better superovulation outcomes. Then, we compared eCG-hCG, IAS-hCG and eCG-IAS-hCG with different dosages in both mouse strains. Significantly increased numbers of oocytes were obtained by using IAS-hCG and eCG-IAS-hCG methods. However, low fertilisation rates (36.3-38.8%) were observed when natural mating was applied. We then confirmed that IVF could dramatically ameliorate the fertilisation rates up to 89.1%. Finally, we performed CRISPR-Cas9 mediated genome editing targeting Scn11a and Kcnh1 loci, and successfully obtained mutant pups using eCG-hCG and IAS-hCG induced zygotes, which were fertilised by either natural mating or IVF. Our results showed that IAS is a promising superovulation reagent, and the efficiency of genome editing is unlikely to be affected by using IAS-induced zygotes.

Identification of chicken-derived scFv against N-glycolylneuraminic acid retrieved from an immune library by phage display.

N- glycolylneuraminic acid (Neu5Gc) is a type of sialic acid, it can be synthesized by a range of mammals except chickens and healthy human. After entering human body, Neu5Gc in foods such as red meat and milk can cause chronic inflammation, thus promoting the development of cancer and related diseases. In this study, we identified a gene sequence of Neu5Gc-specific single-chain variable fragment (ScFv) by phage display from a primary chicken antibodies library. Then the gene sequence was used to express a 29 kDa anti-Neu5Gc ScFv protein as detection probe in competitive inhibition ELISA (IC-ELISA). The linear regression equation of the IC-ELISA was y = 23.12x+33.19 (R = 0.980), and the half-maximal inhibitory concentration (IC50) and the limit of detection (LOD) was 5.333 and 0.66 µg/mL. The mean recovery of the spiked samples was 83.04%, and the intra-assay and inter-assay coefficients of variation (CVs) were both 5.59%. The results suggested that the specific anti-Neu5Gc ScFv is a promising probe for the development of IC-ELISA and test strip in order to detect the presence of Neu5Gc in red meat, milk, and tumor tissues.

Cholecystokinin type 2 receptor in colorectal cancer: diagnostic and therapeutic target.

INTRODUCTION: Cholecystokinin type 2 receptor (CCK2R), which mediates the action of gastrin and cholecystokinin (CCK), has been demonstrated to promote the proliferation of colorectal cancer (CRC). A number of studies showed that CCK2R overexpressed in gastric cancer and pancreatic cancer but few in CRC. The correlation between CCK2R expression and clinicopathological characteristics is also not clear. METHODS: This study investigated CCK2R expression in a wide range of cell lines and clinical CRC samples, and explored expression pattern and prognostic value of CCK2R in relation to clinicopathological parameters. The location and expression levels of CCK2R were measured by immunocytochemical (ICC), qRT-PCR and Western blot. The druggability and antineoplastic effects of CCK2R as a therapeutic target were investigated using an anti-CCK2R targeting recombinant toxin named rCCK8PE38 by CCK-8 assay. RESULTS: Compared with paracarcinoma tissues, tumor samples showed overexpression of CCK2R (p = 0.028) including both CRC tissue and plasma samples, with plasma detection showing a significant indication for CCK2R evaluation. Aberrant expression correlated significantly with histological type (p = 0.032) and p53 status (p < 0.01), and patients with CCK2R overexpression had significantly lower disease-free survival. Application of rCCK8PE38 demonstrated the specificity and druggability of CCK2R as a therapeutic target, providing a strategy for clinical case screening of drugs targeting CCK2R. CONCLUSION: This study highlighted the aberrant expression and clinical correlation of CCK2R and reveals its diagnostic, prognostic and treatment value in CRC. We hypothesize that CCK2R serve as a target for the diagnosis and treatment of this cancer.

A Novel, Rapid, and Simple PMA-qPCR Method for Detection and Counting of Viable Brucella Organisms.

INTRODUCTION: The plate counting method widely used at present to discern viable from non-viable Brucella in the host or cell is time-consuming and laborious. Therefore, it is necessary to establish a rapid, simple method for detecting and counting viable Brucella organisms. MATERIAL AND METHODS: Using propidium monoazide (PMA) to inhibit amplification of DNA from dead Brucella, a novel, rapid PMA-quantitative PCR (PMA-qPCR) detection method for counting viable Brucella was established. The standard recombinant plasmid with the target BCSP31 gene fragment inserted was constructed for drawing a standard curve. The reaction conditions were optimised, and the sensitivity, specificity, and repeatability were analysed. RESULTS: The optimal exposure time and working concentration of PMA were 10 min and 15 µg/mL, respectively. The correlation coefficient (R2) of the standard curve was 0.999. The sensitivity of the method was 103 CFU/mL, moreover, its specificity and repeatability also met the requirements. The concentration of B. suis measured by the PMA-qPCR did not differ significantly from that measured by the plate counting method, and the concentrations of viable bacteria in infected cells determined by the two methods were of the same order of magnitude. CONCLUSION: In this study, a rapid and simple PMA-qPCR counting method for viable Brucella was established, which will facilitate related research.

Characterization of a Novel Interleukin-1 Receptor Antagonist from Sheep (Ovis aries).

Interleukin-1 receptor antagonist (IL-1Ra) is an antagonist of IL-1ß binding IL-1ß receptors but does not induce intracellular responses or signal transduction. In this study, the full-length complementary DNA (cDNA) of the IL-1Ra gene (OaIL-1Ra) was identified from sheep (Ovis aries) using rapid amplification of cDNA ends PCR and submitted to GenBank with the accession number KC425613. The OaIL-1Ra cDNA comprised an open reading frame of 525 bp encoding a protein of 19765.8 Da, a 5'-untranslated region (UTR) of 27 bp, and a 3'-UTR of 676 bp with a poly(A) tail. Recombinant OaIL-1Ra with bioactivity was expressed in a prokaryotic expression system, and a monoclonal antibody against native OaIL-1Ra was prepared. Through Western blot analyses, the OaIL-1Ra protein was widely expressed in lung, heart, spleen, liver, kidney, muscle, intestine, lymphonodi, rumen, and white blood cells, with the highest levels in liver and spleen. The expression of OaIL-1Ra in primary cultured white blood cells of sheep were highly induced in a time-dependent manner when challenged with different bacteria. These results implied that OaIL-1Ra is associated with immune responses during bacterial infections.

Host Prdx6 contributing to the intracellular survival of Brucella suis S2 strain.

BACKGROUND: Brucellosis is a worldwide zoonotic infectious disease that is transmitted in various ways and causes great harm to humans and animals. The brucellosis pathogen is Brucella, which mainly resides in macrophage cells and survives and replicates in host cells. However, the mechanisms underlying Brucella survival in macrophage cells have not been thoroughly elucidated to date. Peroxiredoxin 6 (Prdx6) is a bifunctional protein that shows not only GSH peroxidase activity but also phospholipase A2 activity and plays important roles in combating oxidative damage and regulating apoptosis. RESULTS: Recombinant mouse (Mus musculus) Prdx6 (MmPrdx6) was expressed and purified, and monoclonal antibodies against MmPrdx6 were prepared. Using the Brucella suis S2 strain to infect RAW264.7 murine macrophages, the level of intracellular Prdx6 expression first decreased and later increased following infection. Overexpressing Prdx6 in macrophages resulted in an increase in B. suis S2 strain levels in RAW264.7 cells, while knocking down Prdx6 reduced the S2 levels in cells. CONCLUSIONS: Host Prdx6 can increase the intracellular survival of B. suis S2 strain and plays a role in Brucella infection.

Construction and Activity Analyses of Single Functional Mouse Peroxiredoxin 6 (Prdx6).

INTRODUCTION: Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase activity and phospholipase A2 activity. Previous studies have shown a significant positive correlation between the intracellular survival ability of Brucella and Prdx6. Here, the Prdx6 enzyme with a single activity was constructed to facilitate study of the relationship between the single function of Prdx6 and Brucella infection. MATERIAL AND METHODS: The target open reading frame (ORF) DNAs of Prdx6 with a single active centre were prepared using gene splicing by overlap extension PCR (SOE-PCR), and the recombinant eukaryotic expression plasmids inserted by Prdx6 with the single activity centre were constructed and transfected into murine Raw264.7 macrophages. The glutathione peroxidase activity and phospholipase A2 activity of the constructed Prdx6 were examined. RESULTS: The core centres (Ser32 and Cys47) of Prdx6 were successfully mutated by changing the 94th nucleotide from T to G and the 140th nucleotide from G to C in the two enzyme activity cores, respectively. The constructed recombinant plasmids of Prdx6 with the single active centre were transfected into murine macrophages showing the expected single functional enzyme activity, which MJ33 or mercaptosuccinate inhibitors were able to inhibit. CONCLUSION: The constructed mutants of Prdx6 with the single activity cores will be a benefit to further study of the biological function of Prdx6 with different enzyme activity.

Cloning and Differential Expression Analyses of Cdc42 from Sheep.

INTRODUCTION: Serological diagnosis of brucellosis is still a great challenge due to the infeasibility of discriminating infected animals from vaccinated ones, so it is necessary to search for diagnostic biomarkers for differential diagnosis of brucellosis. MATERIAL AND METHODS: Cell division cycle 42 (Cdc42) from sheep (Ovis aries) (OaCdc42) was cloned by rapid amplification of cDNA ends (RACE), and then tissue distribution and differential expression levels of OaCdc42 mRNA between infected and vaccinated sheep were analysed by RT-qPCR. RESULTS: The full-length cDNA of OaCdc42 was 1,609 bp containing an open reading frame (ORF) of 576 bp. OaCdc42 mRNAs were detected in the heart, liver, spleen, lung, kidneys, rumen, small intestine, skeletal muscles, and buffy coat, and the highest expression was detected in the small intestine. Compared to the control, the levels of OaCdc42 mRNA from sheep infected with Brucella melitensis or sheep vaccinated with Brucella suis S2 was significantly different (P < 0.01) after 40 and 30 days post-inoculation, respectively. However, the expression of OaCdc42 mRNA was significantly different between vaccinated and infected sheep (P < 0.05 or P < 0.01) on days: 14, 30, and 60 post-inoculation, whereas no significant difference (P > 0.05) was noted 40 days post-inoculation. Moreover, the expression of OaCdc42 from both infected and vaccinated sheep showed irregularity. CONCLUSION: OaCdc42 is not a good potential diagnostic biomarker for differential diagnosis of brucellosis in sheep.

An efficient scheme for purification of a novel recombinant immunotoxin, rCCK8PE38, for anti-tumour experiments.

rCCK8PE38 is a novel immunotoxin that targets choleystokinin B receptor, which is over-expressed in some tumor tissues. Although we constructed a prokaryotic expression vector to express rCCK8PE38 in our laboratory, thorough purification was necessary to quantitatively assess its anti-tumor effect. In this study, we established a purification protocol to obtain rCCK8PE38 with high purity from E. coli. Three different types of chromatography, hydrophobic chromatography, ion exchange chromatography and size exclusion chromatography, were used in combination. The purification technological parameters of each chromatography type were optimized. The whole process of purification was arranged to minimize the purification steps and achieve purity and bioactivity. Finally, through this optimized scheme, we obtained a recombinant protein with a purity of >95%; then, the protein was stored at -80°C after lyophilization. The purified protein was used in a tumor inhibition experiment and was effective in killing tumor cells that over-expressed choleystokinin B receptor. The results of this study may provide some valuable information about protein purification and lay the foundation for further clinical experiments with rCCK8PE38.

Molecular characterization and differential expression analysis of interleukin 1ß from Ovis aries.

The interleukin-1 family is an important component of the innate immune system and plays an important role in regulating immune responses on the invasion of intracellular parasites in the acquired immune system. Interleukin 1ß (IL-1ß) is one of the members of the IL-1 family that predominantly activates downstream signaling pathways to play immunological functions of stimulating T and B lymphocyte activation and promoting the various syntheses of inflammatory substances in conjunction with other cytokines. Here, a full-length IL-1ß cDNA (OaIL-1ß) of sheep (Ovis aries) was cloned using rapid amplification of cDNA ends (RACE), which consists of 1494 bp and contains a 5'-UTR region with a length of 83 bp, a complete ORF of 801 bp in length, and a 3'-UTR region with a length of 642 bp. Recombinant protein OaIL-1ß was expressed and purified, and the monoclonal antibody against IL-1ß of sheep is prepared. Western blotting results showed that the sheep IL-1ß protein was detected in the heart, liver, lung, kidney, stomach, intestine, muscle, lymph nodes and leukocytes with the highest expression in the muscle and the lowest expression in the lung. Different bacteria treating sheep white blood cells induced differential expression of OaIL-1ß. Compared with the normal sheep, OaIL-1ß in the buffy coat was differentially expressed in the Brucella melitensis-challenged group and the B. suis S2 strain-inoculated group. However, whether IL-1ß may be considered as a molecular biomarker for differing Brucella-infected animals from brucellosis-vaccinated animals or not need to be further studied.

Clinical targeting recombinant immunotoxins for cancer therapy.

Recombinant immunotoxins (RITs) are proteins that contain a toxin fused to an antibody or small molecules and are constructed by the genetic engineering technique. RITs can bind to and be internalized by cells and kill cancerous or non-cancerous cells by inhibiting protein synthesis. A wide variety of RITs have been tested against different cancers in cell culture, xenograft models, and human patients during the past several decades. RITs have shown activity in therapy of several kinds of cancers, but different levels of side effects, mainly related to vascular leak syndrome, were also observed in the treated patients. High immunogenicity of RITs limited their long-term or repeat applications in clinical cases. Recent advances in the design of immunotoxins, such as humanization of antibody fragment, PEGylation, and modification of human B- and T-cell epitopes, are overcoming the above mentioned problems, which predict the use of these immunotoxins as a potential therapeutic method to treat cancer patients.

Exploration of the Main Sites for the Transformation of Normal Prion Protein (PrPC) into Pathogenic Prion Protein (PrPsc).

INTRODUCTION: The functions and mechanisms of prion proteins (PrPC) are currently unknown, but most experts believe that deformed or pathogenic prion proteins (PrPSc) originate from PrPC, and that there may be plural main sites for the conversion of normal PrPC into PrPSc. In order to better understand the mechanism of PrPC transformation to PrPSc, the most important step is to determine the replacement or substitution site. MATERIAL AND METHODS: BALB/c mice were challenged with prion RML strain and from 90 days post-challenge (dpc) mice were sacrificed weekly until all of them had been at 160 dpc. The ultra-structure and pathological changes of the brain of experimental mice were observed and recorded by transmission electron microscopy. RESULTS: There were a large number of pathogen-like particles aggregated in the myelin sheath of the brain nerves, followed by delamination, hyperplasia, swelling, disintegration, phagocytic vacuolation, and other pathological lesions in the myelin sheath. The aggregated particles did not overflow from the myelin in unstained samples. The phenomenon of particle aggregation persisted all through the disease course, and was the earliest observed pathological change. CONCLUSION: It was deduced that the myelin sheath and lipid rafts in brain nerves, including axons and dendrites, were the main sites for the conversion of PrPC to PrPSc, and the PrPSc should be formed directly by the conversion of protein conformation without the involvement of nucleic acids.

Full-Length cDNA Cloning, Molecular Characterization and Differential Expression Analysis of Lysophospholipase I from Ovis aries.

Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5'-untranslated region (UTR) of 24 bp, a 3'-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future.

Characterization of a highly virulent and antimicrobial-resistant Acinetobacter baumannii strain isolated from diseased chicks in China.

Poultry husbandry is a very important aspect of the agricultural economy in China. However, chicks are often susceptible to infectious disease microorganisms, such as bacteria, viruses and parasites, causing large economic losses in recent years. In the present study, we isolated an Acinetobacter baumannii strain, CCGGD201101, from diseased chicks in the Jilin Province of China. Regression analyses of virulence and LD50 tests conducted using healthy chicks confirmed that A. baumannii CCGGD201101, with an LD50 of 1.81 (±0.11) × 10(4) CFU, was more virulent than A. baumannii ATCC17978, with an LD50 of 1.73 (±0.13) × 10(7) CFU. Moreover, TEM examination showed that the pili of A. baumannii CCGGD201101 were different from those of ATCC17978. Antibiotic sensitivity analyses showed that A. baumannii CCGGD201101 was sensitive to rifampicin but resistant to most other antibiotics. These results imply that A. baumannii strain CCGGD201101 had both virulence enhancement and antibiotic resistance characteristics, which are beneficial for A. baumannii survival under adverse conditions and enhance fitness and invasiveness in the host. A. baumannii CCGGD20101, with its high virulence and antimicrobial resistance, may be one of the pathogens causing death of diseased chicks.

Characterization of surface antigen protein 1 (SurA1) from Acinetobacter baumannii and its role in virulence and fitness.

Acinetobacter baumannii is a Gram-negative bacillus that causes nosocomial infections, such as bacteremia, pneumonia, and meningitis and urinary tract and wound infections. In the present study, the surface antigen protein 1 (SurA1) gene of A. baumannii strain CCGGD201101 was identified, cloned and expressed, and then its roles in fitness and virulence were investigated. Virulence was observed in the human lung cancer cell lines A549 and HEp-2 at one week after treatment with recombinant SurA1. One isogenic SurA1 knock-out strain, GR0015, which was derived from the A. baumannii strain CCGGD201101 isolated from diseased chicks in a previous study, highlighted the effect of SurA1 on fitness and growth. Its growth rate in LB broth and killing activity in human sera were significantly decreased compared with strain CCGGD201101. In the Galleria mellonella insect model, the isogenic SurA1 knock-out strain exhibited a lower survival rate and decreased dissemination. These results suggest that SurA1 plays an important role in the fitness and virulence of A. baumannii.

Preparation of a Specific ssDNA Aptamer for Brevetoxin-2 Using SELEX.

The existing assays for detecting brevetoxin (BTX) depend on expensive equipment with a professional operator or on an antibody with limited stability, which requires complex processes, a high cost, and a considerable amount of time. The development of an alternative detection probe is another promising research direction. This paper reports the use of aptamers binding to BTX-2 in an analytical assay using the systematic evolution of ligands by exponential enrichment (SELEX). After 12 rounds of selection, the secondary structures of 25 sequences were predicted. Compared to other aptamers, Bap5 has relatively high affinity with the lowest dissociation constant of 4.83 µM, and IC50 is 73.81 ng mL-1. A good linear regression formula of y = 30.688x - 7.329 with a coefficient correlation of R2 = 0.9798 was obtained using a biotin-avidin ELISA. Moreover, there is no cross-reaction with the detected marine toxins, except for BTX-2. Thus, Bap5 has potential to detect BTX-2 in shellfish in the future as a substitute for the recognition probe.

Generation of Internal-Image Functional Aptamers of Okadaic Acid via Magnetic-Bead SELEX.

Okadaic acid (OA) is produced by Dinophysis and Prorocentrum dinoflagellates and primarily accumulates in bivalves, and this toxin has harmful effects on consumers and operators. In this work, we first report the use of aptamers as novel non-toxic probes capable of binding to a monoclonal antibody against OA (OA-mAb). Aptamers that mimic the OA toxin with high affinity and selectivity were generated by the magnetic bead-assisted systematic evolution of ligands by exponential enrichment (SELEX) strategy. After 12 selection rounds, cloning, sequencing and enzyme-linked immunosorbent assay (ELISA) analysis, four candidate aptamers (O24, O31, O39, O40) were selected that showed high affinity and specificity for OA-mAb. The affinity constants of O24, O31, O39 and O40 were 8.3 × 108 M(-1), 1.47 × 108 M(-1), 1.23 × 108 M(-1) and 1.05 × 108 M(-1), respectively. Indirect competitive ELISA was employed to determine the internal-image function of the aptamers. The results reveal that O31 has a similar competitive function as free OA toxin, whereas the other three aptamers did not bear the necessary internal-image function. Based on the derivation of the curvilinear equation for OA/O31, the equation that defined the relationship between the OA toxin content and O31 was Y = 2.185X - 1.78. The IC50 of O31 was 3.39 ng·mL(-1), which was close to the value predicted by the OA ELISA (IC50 = 4.4 ng·mL(-1)); the IC10 was 0.33 ng·mL(-1). The above data provides strong evidence that internal-image functional aptamers could be applicable as novel probes in a non-toxic assay.