Inter-assay Coefficients Of Variation Research Articles

Development of an enzyme-linked immunosorbent assay for determining FSH plasma concentrations in green sea turtle (Chelonia mydas), using recombinant gonadotropins

Follicle-stimulating hormone (FSH) is involved in the regulation of essential reproductive processes such as gametogenesis and follicular growth. There are presently no immunoassays for measuring FSH in turtles. Recently we produced green sea turtle (Chelonia mydas) recombinant (r) FSH as a single-chain polypeptide using the methylotrophic yeast Pichia pastoris expression system, and polyclonal antibodies for the recombinant FSH. In this work we developed a competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of FSH concentrations in plasma samples from green sea turtles. We used the rFSHβα for standard, rFSHβ for coating and a cmFSHβ antibody. The sensitivity of the assay was 0.13 ng/ml and the intra-assay and inter-assay coefficients of variation were 5.54% and 13.52% respectively. Parallelism was observed between the linearized FSH standard curves and the corresponding serial dilutions of green sea turtle plasma samples. We also observed parallelism between the linearized standard and serial dilutions of plasma samples from the loggerhead sea turtle (Caretta caretta), hawksbill sea turtle (Eretmochelys imbricate), and African softshell turtle (Trionyx triunguis). The ELISA was used to study the FSH plasma concentrations during the reproductive cycles and was compared to hormonal steroid concentrations (Testosterone, Estradiol and Progesterone). This revealed a positive correlation between FSH and estradiol concentrations in females; estradiol concentrations were increased immediately after FSH elevation. In addition, nested females presented an increase in FSH concentrations prior to progesterone elevation in January to April, slightly before egg laying. This ELISA will increase our understanding of gonadotropin functions, and their effects on reproduction in the green sea turtle.

Development of SYBR Green I-based quantitative PCR assay for identification of porcine circovirus 1, 2 and 3

Porcine Circovirus (PCV) includes Porcine Circovirus 1(PCV1), Porcine Circovirus 2 (PCV2) and Porcine Circovirus 3 (PCV3). In recent years, co-infection exists between PCV1, PCV2 and PCV3 serotypes. Therefore, it is particularly necessary to establish a fast, specific and sensitive SYBR Green I real-time quantitative PCR detection method for PCV1, PCV2 and PCV3. In this experiment, specific primers were selected and the reaction conditions were optimized. A real-time quantitative PCR identification method was established. The results showed the detection limits of this assay were 40.3 copies/μl for PCV1, 25.2 copies/μl for PCV2 and22.4 copies/ μl for PCV3. There was no cross-reactivity with swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV). The intra-assay and inter-assay coefficients of variation were less than 1%. The test results of 100 PCV suspected positive samples revealed that the PCV1, PCV2 and PCV3 singular infection rate was 10% (10/100), 64% (64/100) and 52% (52/100), respectively. The PCV1 and PCV2 co-infection rate was 8% (8/100), the PCV1 and PCV3 co-infection rate was 7% (7/100), the PCV2 and PCV3 co-infection rate was 26% (26/100), and the PCV1, PCV2 and PCV3 co-infection rate was 7% (7/100). This method has good specificity, sensitivity and stability. It provides a promising tool for rapid differential detection of PCV1, PCV2 and PCV3.

Evaluation of the fully automated LIAISON®XL chemiluminescence analyzer for QuantiFERON®-CMV testing in transplant recipients.

Monitoring CMV-specific cell-mediated immunity by the QuantiFERON®-CMV (QF-CMV) may be useful in predicting the risk of CMV infection in transplant recipients (TR). As the QuantiFERON-Tuberculosis (QFT®-Plus) became available on the fully automated LIAISON®XL chemiluminescence (CLIA) analyzer, we evaluated the performance of the QF-CMV on the LIAISON®XL analyzer using the QuantiFERON®-TB Gold Plus reagent. Between 2018 and 2022, 81 samples from TR were collected at the Department of Virology of Limoges Hospital, France. Whole blood was collected into each of the three QF-CMV collection tubes: a CMV-antigen tube (QF-Ag), a mitogen tube (QF-Mg) (positive control), and a nil tube (negative control). The QF-CMV was performed on the LIAISON®XL analyzer, and results were compared with those obtained by conventional microplate ELISA. Intra- and inter-assay coefficients of variation were inferior to 20%. No inter-sample contamination was found (p=0.366). The level of concordance between CLIA and the commonly used ELISA method was 88.9%. Positive and negative percent agreements were 92.3% and 85.7%, respectively, with a very good agreement between assays (κ=0.818). Most discordances were due to indeterminate- or negative-ELISA/positive-CLIA results (most of ELISA results were borderline). Linear regression analyses demonstrated a strong correlation between both assays (QF-Ag Pearson's r=0.978, QF-Mg Pearson's r=0.963). No significant difference was observed in median QF-CMV values between both assays (QF-Ag p=0.776; QF-Mg p=0.853; Mann-Whitney U test). The Bland-Altman plots showed a minor difference in IFN-γ release (QF-Ag -0.069 IU/ml, 95% limits of agreement (LoA): -1.589; 1.451; QF-Mg0.190IU/ml, 95% LoA: -2.070; 2.450). Automated QF-CMV with CLIA is comparable to QF-CMV performed by ELISA with a presumably higher sensitivity for IFN-γ detection that may result in the conversion of samples close to the ELISA cut-off into positive results. Moreover, the use of a random-access analyzer allows to optimize the follow-up of TR.

Triplex Crystal Digital PCR for the Detection and Differentiation of the Wild-Type Strain and the MGF505-2R and I177L Gene-Deleted Strain of African Swine Fever Virus.

African swine fever (ASF) is a severe and highly contagious viral disease that affects domestic pigs and wild boars, characterized by a high fever and internal bleeding. The disease is caused by African swine fever virus (ASFV), which is prevalent worldwide and has led to significant economic losses in the global pig industry. In this study, three pairs of specific primers and TaqMan probes were designed for the ASFV B646L, MGF505-2R and I177L genes. After optimizing the reaction conditions of the annealing temperature, primer concentration and probe concentration, triplex crystal digital PCR (cdPCR) and triplex real-time quantitative PCR (qPCR) were developed for the detection and differentiation of the wild-type ASFV strain and the MGF505-2R and/or I177L gene-deleted ASFV strains. The results indicate that both triplex cdPCR and triplex qPCR were highly specific, sensitive and repeatable. The assays could detect only the B646L, MGF505-2R and I177L genes, without cross-reaction with other swine viruses (i.e., PRRSV, CSFV, PCV2, PCV3, PEDV, PDCoV and PRV). The limit of detection (LOD) of triplex cdPCR was 12 copies/reaction, and the LOD of triplex qPCR was 500 copies/reaction. The intra-assay and inter-assay coefficients of variation (CVs) for repeatability and reproducibility were less than 2.7% for triplex cdPCR and less than 1.8% for triplex qPCR. A total of 1510 clinical tissue samples were tested with both methods, and the positivity rates of ASFV were 14.17% (214/1510) with triplex cdPCR and 12.98% (196/1510) with triplex qPCR, with a coincidence rate of 98.81% between the two methods. The positivity rate for the MGF505-2R gene-deleted ASFV strains was 0.33% (5/1510), and no I177L gene-deleted ASFV strain was found. The results indicate that triplex cdPCR and triplex qPCR developed in this study can provide rapid, sensitive and accurate methods for the detection and differentiation of the ASFV B646L, MGF505-2R and I177L genes.

Development of a Quadruplex RT-qPCR for the Detection of Porcine Rotaviruses and the Phylogenetic Analysis of Porcine RVH in China.

Rotavirus A species (RVA), RVB, RVC, and RVH are four species of rotaviruses (RVs) that are prevalent in pig herds, and co-infections occur frequently. In this study, a quadruplex real-time quantitative RT-PCR (RT-qPCR) for the simultaneous detection of four porcine RVs was developed by designing specific primers and probes based on the VP6 gene of RVA, RVB, RVC, and RVH, respectively. The method showed high specificity and could only detect RVA, RVB, RVC, and RVH, without cross-reaction with other porcine viruses; showed excellent sensitivity, with a limit of detection (LOD) of 1.5 copies/µL for each virus; showed good repeatability, with intra-assay coefficients of variation (CVs) of 0.15-1.14% and inter-assay CVs of 0.07-0.96%. A total of 1447 clinical fecal samples from Guangxi province in China were tested using the developed quadruplex RT-qPCR. The results showed that RVA (42.71%, 618/1447), RVB (26.95%, 390/1447), RVC (42.92%, 621/1447), and RVH (13.68%, 198/1447) were simultaneously circulating in the pig herds, and the co-infection rate of different species of rotaviruses was found to be up to 44.01% (579/1447). The clinical samples were also detected using one previously reported method, and the coincidence rate of the detection results using two methods was more than 99.65%. The phylogenetic tree based on the VP6 gene sequences of RVH revealed that the porcine RVH strains from Guangxi province belonged to the genotype I5, which was closely related to Japanese and Vietnamese strains. In summary, an efficient, sensitive, and accurate method for the detection and differentiation of RVA, RVB, RVC, and RVH was developed and applied to investigate the prevalence of porcine RVs in Guangxi province, China. This study is the first to report the prevalence of porcine RVH in China.

Anti-Treponema pallidum IgA response as a potential diagnostic marker of syphilis

ObjectivesSerological tests for syphilis detect mainly total Ig, IgM or IgG antibodies. We aimed to evaluate the specific IgA response in syphilis patients according to disease stage. MethodsA serum IgA-enzyme immunoassay was developed using commercially available microplates coated with recombinant treponemal antigens and an anti-IgA-conjugate. To define a cut-off, we used 91 syphilis positive and 136 negative sera previously defined by the rapid plasma reagin and the Treponema pallidum particle agglutination results. Then we determined the intra- and inter-assay precisions, diagnostic sensitivity according to the clinical stage (in 66, 55 and 42 sera from primary, secondary and latent syphilis patients, respectively) and specificity (in 211 sera from people with conditions different to syphilis). IgA values were further measured in 71 sera from patients with previously treated syphilis. ResultsThe newly developed IgA-enzyme immunoassay showed a good discrimination between negative and positive samples with intra- and inter-assay variation coefficients <20%. The sensitivity was 80.3% (95% CI, 70.0–90.6), 100.0% (95% CI, 99.1–100.0) and 95.2% (95% CI, 87.6–100.0) in primary, secondary and latent syphilis, respectively, and the specificity was 98.1% (95% CI, 96.0–100.0). Further, IgA values were negative in 61.3% (38/62) of patients with previously treated syphilis. DiscussionOur findings suggest serum IgA as a sensitive and specific marker of syphilis and its detection could be used as a screening assay for active infection. Further evaluation is needed in prospective longitudinal field studies.

Okadaic Acid Detection through a Rapid and Sensitive Amplified Luminescent Proximity Homogeneous Assay.

Okadaic acid (OA), a marine biotoxin produced by microalgae, poses a significant threat to mariculture, seafood safety, and human health. The establishment of a novel, highly sensitive detection method for OA would have significant practical and scientific implications. Therefore, the purpose of this study was to develop an innovative approach for OA detection. A competitive amplified luminescent proximity homogeneous assay (AlphaLISA) was developed using the principle of specific antigen-antibody binding based on the energy transfer between chemiluminescent microspheres. The method was non-washable, sensitive, and rapid, which could detect 2 × 10-2-200 ng/mL of OA within 15 min, and the detection limit was 4.55 × 10-3 ng/mL. The average intra- and inter-assay coefficients of variation were 2.54% and 6.26%, respectively. Detection of the actual sample results exhibited a good correlation with high-performance liquid chromatography. In conclusion, a simple, rapid, sensitive, and accurate AlphaLISA method was established for detecting OA and is expected to significantly contribute to marine biotoxin research.

Multiplex digital PCR: a superior technique to qPCR for the simultaneous detection of duck Tembusu virus, duck circovirus, and new duck reovirus.

Duck Tembusu virus (DTMUV), duck circovirus (DuCV), and new duck reovirus (NDRV) have seriously hindered the development of the poultry industry in China. To detect the three pathogens simultaneously, a multiplex digital PCR (dPCR) was developed and compared with multiplex qPCR in this study. The multiplex dPCR was able to specifically detect DTMUV, DuCV, and NDRV but not amplify Muscovy duck reovirus (MDRV), Muscovy duck parvovirus (MDPV), goose parvovirus (GPV), H4 avian influenza virus (H4 AIV), H6 avian influenza virus (H6 AIV), and Newcastle disease virus (NDV). The standard curves showed excellent linearity in multiplex dPCR and qPCR and were positively correlated. The sensitivity results showed that the lowest detection limit of multiplex dPCR was 1.3 copies/μL, which was 10 times higher than that of multiplex qPCR. The reproducibility results showed that the intra- and interassay coefficients of variation were 0.06-1.94%. A total of 173 clinical samples were tested to assess the usefulness of the method; the positive detection rates for DTMUV, DuCV, and NDRV were 18.5, 29.5, and 14.5%, respectively, which were approximately 4% higher than those of multiplex qPCR, and the kappa values for the clinical detection results of multiplex dPCR and qPCR were 0.85, 0.89, and 0.86, indicating that the two methods were in excellent agreement.

Measuring ADAMTS-13 activity to diagnose thrombotic thrombocytopenic purpura: a novel, fast fiber-optic surface plasmon resonance immunoassay

BackgroundThrombotic thrombocytopenic purpura (TTP) is characterized by severe ADAMTS-13 activity deficiency (<10%). Diagnostic testing is challenging because of unavailability, high cost, and expert technician requirement of ADAMTS-13 enzyme assays. Cost-effective, automated fiber-optic surface plasmon resonance (FO-SPR) platforms show potential for developing diagnostic tests. Yet, FO-SPR has never been explored to measure enzymatic activities. ObjectivesTo develop an easy-to-use ADAMTS-13 activity assay utilizing optical fibers to rapidly diagnose TTP. MethodsThe ADAMTS-13 activity assay was designed and optimized using FO-SPR technology based on a previously described enzyme-linked immunosorbent assay setup. A calibration curve was generated to quantify ADAMTS-13 activity in plasma of healthy donors and patients with acute immune-mediated TTP (iTTP), hemolytic uremic syndrome, or sepsis. ADAMTS-13 activity data from FO-SPR and fluorescence resonance energy transfer-based strategies (FRETS)-VWF73 reference assays were compared. ResultsAfter initial assay development, optimization improved read-out magnitude and signal-to-noise ratio and reduced variation. Further characterization demonstrated a detection limit (6.8%) and inter-assay variation (Coefficient of variation, 7.2%) that showed good analytical sensitivity and repeatability. From diverse plasma samples, only plasma from patients with acute iTTP showed ADAMTS-13 activities below 10%. Strong Pearson correlation (r = 0.854) between FO-SPR and reference FRETS-VWF73 assays were observed for all measured samples. ConclusionsA fast ADAMTS-13 activity assay was designed onto automated FO-SPR technology. Optimization resulted in sensitive ADAMTS-13 activity measurements with a detection limit enabling clinical diagnosis of TTP within 3 hours. The FO-SPR assay proved strong correlation with the reference FRETS-VWF73 assay. For the first time, this assay demonstrated the capacity of FO-SPR technology to measure enzymatic activity in pre-clinical context.

Determination of the Elemental Iodine in Human Breast Milk by Inductively Coupled Plasma mass Spectrometry.

Adequate iodine status in lactating women is defined by a maternal median urinary iodine concentration (UIC) ≧ 100µg/L. However, the above-mentioned criterion does not account for the secretion of iodine into breast milk and could not truly reflect the amount of iodine delivered to the infants. Measuring breast milk median iodine concentration (BMIC) is crucial, but the method to measure BMIC has not been developed and validated in Taiwan. We adopted the ammonia dilution method without prior sample digestion to measure BMIC by inductively coupled plasma mass spectrometry (ICP-MS). Samples and iodate calibrators were prepared into an aqueous solution containing Triton X-100, 0.5% ammonia solution, and tellurium (128Te) as the internal standard. Precision, accuracy, serial dilution, and recovery tests were performed for method validation. The range of intra-assay and inter-assay coefficient of variation for the four human breast milk samples with different iodine concentrations were 3.2-4.7% and 2.3-5.5%, respectively. The standard NIST 1549 milk powder was prepared into three different concentrations of 50µg/L, 100µg/L, and 200µg/L to assess the accuracy; the bias was < 5%. A recovery of 95-105% was achieved for four human breast milk samples spiked with sodium iodide solution. The serial dilution test confirmed linearity up to 0.998. The limit for detection and quantification was 0.78µg/L and 2.34µg/L, respectively. The results of the current study confirmed that this ICP-MS method is accurate and reliable in measuring BMIC.

Production of virus-like particles of porcine circovirus 2 in baculovirus expression system and its application for antibody detection

BackgroundPorcine circovirus 2 (PCV-2) is one of the pathogens that leads to a growing and persistent threat in pigs. Thus, the development of serological detection methods for PCV-2 is of great necessity for clinical diagnosis as well as epidemiological investigations. This study aimed to establish an indirect enzyme-linked immunosorbent assay (ELISA) to examine antibodies against PCV-2 based on virus-like particles (VLPs).ResultsRecombinant PCV-2 Cap protein was expressed in the baculovirus-insect cells system and PCV-2 VLPs were observed over transmission electron microscopy (TEM). The PCV-2 VLPs were shown to have good immunogenicity in mice and stimulated a high level of PCV-2 antibody titers. Using PCV-2 VLPs as coating antigen, the indirect ELISA can detect PCV-2 antibodies in animals with diagnostic sensitivity and specificity of 98.33% and 93.33% compared to immunofluorescence assay (IFA), respectively. The intra- and inter-assay coefficient variations (CVs) were < 10% in a batch, and < 15% in different batches, indicating good repeatability. There was no cross-reaction of this ELISA with antibodies against other porcine viruses. A total of 170 serum samples collected from different pig farms in China were tested for PCV-2 antibodies, and 151 (88.8%) samples were PCV-2 antibody positive.ConclusionOur findings suggest that this ELISA was rapid, specific, and reproducible and can be used for large-scale serological investigations of PCV-2 antibodies in pigs.

Absolute quantification of neuromelanin in formalin-fixed human brains using absorbance spectrophotometry.

Parkinson's disease is characterised by a visual, preferential degeneration of the pigmented neurons in the substantia nigra. These neurons are pigmented by neuromelanin which decreases in Parkinson's disease. Not much is known about NM as it is difficult to study and quantify, primarily due to its insolubility in most solvents except alkali. Neuromelanin quantification could progress the development of biomarkers for prodromal Parkinson's disease and provide insights into the presently unclear role of neuromelanin in Parkinson's disease aetiology. Light microscopy with stereology can visualise pigmented neurons, but it cannot quantify neuromelanin concentrations. Absolute neuromelanin quantification using absorbance spectrophotometry is reported in the literature, but the methodology is dated and only works with fresh-frozen tissue. We have developed a quantification protocol to overcome these issues. The protocol involves breakdown of fixed tissue, dissolving the tissue neuromelanin in sodium hydroxide, and measuring the solution's 350 nm absorbance. Up to 100 brain samples can be analysed in parallel, using as little as 2 mg of tissue per sample. We used synthetic neuromelanin to construct the calibration curve rather than substantia nigra neuromelanin. Our protocol enzymatically synthesises neuromelanin from dopamine and L-cysteine followed by high-heat ageing. This protocol enables successful lysis of the fixed substantia nigra tissue and quantification in three brains, with neuromelanin concentrations ranging from 0.23-0.55 μg/mg tissue. Quantification was highly reproducible with an interassay coefficient of variation of 6.75% (n = 5). The absorbance spectra and elemental composition of the aged synthetic neuromelanin and substantia nigra neuromelanin show excellent overlap. Our protocol can robustly and reliably measure the absolute concentration of neuromelanin in formalin-fixed substantia nigra tissue. This will enable us to study how different factors affect neuromelanin and provide the basis for further development of Parkinson's disease biomarkers and further research into neuromelanin's role in the brain.

Construction of a one-step multiplex real-time PCR assay for the detection of serogroups A, B, and E of Pasteurella multocida associated with bovine pasteurellosis.

Bovine pasteurellosis, caused by serogroups A, B, and E of Pasteurella multocida (Pm), is mainly manifested as bovine respiratory disease (BRD) and hemorrhagic septicemia (HS). The disease has caused a great economic loss for the cattle industry globally. Therefore, identifying the Pm serogroups is critical for optimal diagnosis and subsequent clinical treatment and even epidemiological studies. In this study, a one-step multiplex real-time PCR assay was established. Three pairs of specific primers were prepared to detect the highly conserved genomic regions of serogroups A (HyaD), B (bcbD), and E (ecbJ) of Pm, respectively. The results depicted that the method had no cross-reaction with other bovine pathogens (Mannheimia hemolytica, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Salmonella Dublin, Mycobacterium paratuberculosis, infectious bovine rhinotracheitis virus, and Mycoplasma bovis). The linear range (107 to 102 copies/μL) showed the R2 values for serogroups A, B, and E of Pm as 0.9975, 0.9964, and 0.996, respectively. The multiplex real-time PCR efficiency was 90.30%, 90.72%, and 90.57% for CartA, CartB, and CartE, respectively. The sensitivity result showed that the serogroups A, B, and E of Pm could be detected to be as low as 10 copies/μL. The repeatability result clarified that an intra-assay and an inter-assay coefficient of variation of serogroups A, B, and E of Pm was < 2%. For the clinical samples, the detection rate was higher than the OIE-recommended ordinary PCR. Overall, the established one-step multiplex real-time PCR assay may be a valuable tool for the rapid and early detection of the serogroups A, B, and E of Pm with high specificity and sensitivity.

High-Throughput CD36 Phenotyping on Human Platelets Based on Sandwich ELISA and Mutant Gene Analysis

Background: CD36 deficiency is closely associated with fetal/neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness, and other hemorrhage disorders, particularly in Asian and African populations. There is a clinical need for rapid and high-throughput methods of platelet CD36 (pCD36) phenotyping to improve the availability of CD36 typing of donors and assist clinical blood transfusions for patients with anti-CD36 antibodies. Such methods can also support the establishment of databases of pCD36-negative phenotypes. Study Design and Methods: A sandwich enzyme-linked immunosorbent assay (ELISA) for CD36 phenotyping of human platelets was developed using anti-CD36 monoclonal antibodies. The reliability of the assay was evaluated by calculating the intra-assay and inter-assay coefficients of variation (CV). A total of 1,691 anticoagulant whole blood samples from healthy blood donors were randomly selected. PCD36 expression was measured using a sandwich ELISA. PCD36 deficiency was confirmed by flow cytometry (FC). Mutations underlying pCD36 deficiency were identified using polymerase chain reaction sequence-based typing (PCR-SBT). Results: The sandwich ELISA for pCD36 phenotyping had high reliability (intra-assay CV, 2.1–4.8%; inter-assay CV, 2.3–5.2%). The sandwich ELISA was used to screen for CD36 expression on platelets isolated from 1,691 healthy blood donors. Of these, 36 samples were pCD36-negative. FC demonstrated absence of CD36 expression on monocytes in three of the 36 cases. In the present study population, the frequency of CD36 deficiency was 2.13% (36/1,691), of which 0.18% (3/1,691) was type I deficiency and 1.95% (33/1,691) was type II deficiency. In addition, we used PCR-SBT to characterize the gene mutations in exons 3–14 of the CD36 gene in 27 cases of CD36 deficiency and discovered 10 types of mutations in 13 pCD36-negative samples. Conclusion: The present study describes the development and characterization of a highly reliable sandwich ELISA for high-throughput screening for pCD36 expression. This novel method is feasible for clinical applications and provides a useful tool for the establishment of databases of pCD36-negative phenotype donors.

Development and Application of a Nanobody-Based Competitive ELISA for Detecting Antibodies against Hepatitis E Virus from Humans and Domestic Animals.

Hepatitis E virus (HEV) is a zoonotic pathogen that is widespread worldwide. At present, most enzyme-linked immunosorbent assay (ELISA) kits only detect antibodies against human HEV. In this study, a nanobody-horseradish peroxidase (HRP) fusion protein-based competitive ELISA (cELISA) with more convenience and spectral characteristics for HEV antibody detection was developed and used to detect HEV IgG in various species. First, 6 anti-swine HEV capsid protein nanobodies were screened using phage display technology from an immunized Bactrian camel. Then, HEV-Nb67-HRP fusions were expressed and used as a probe for developing a cELISA. The cutoff value of the cELISA was 17.8%, and there was no cross-reaction with other anti-swine virus antibodies, suggesting that the cELISA had good specificity. The intra-assay and interassay coefficients of variation (CVs) were 1.33 to 5.06% and 1.52 to 6.84%, respectively. The cELISA and Western blot showed a higher coincidence rate (97.14%, kappa value = 0.927) than cELISA and indirect ELISA (95.00%, kappa value = 0.876) in clinical swine serum samples. Finally, the seroprevalence of HEV IgG in humans, pigs, rabbits, cows, and goats was 30.67%, 19.26%, 8.75%, 27.59%, and 18.08%, respectively, suggesting that cELISA may have a broader scale for mammalian HEV antibody detection. These results suggest that the newly developed cELISA was rapid, low-cost, reliable, and useful for the serological evaluation of current HEV. IMPORTANCE HEV is thought to be a zoonotic infection and is widespread worldwide; it is beneficial to establish a more convenient and spectral method for HEV antibody detection. In this study, a convenient, time-saving, reproducible, highly sensitive, specific, and novel nanobody-based cELISA was developed and can be used to detect IgG antibodies against mammalian HEV. It provides a new technique for serological evaluation and ELISA-based diagnosis of HEV infection.

A specific and sensitive droplet digital polymerase chain reaction assay for the detection of tilapia lake virus in fish tissue and environmental samples.

Tilapia lake virus (TiLV) causes high mortality in farmed and wild tilapia in various countries. We developed a highly specific and sensitive droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify TiLV. The ddPCR assay could detect the virus at a lower threshold than the reverse transcription-quantitative polymerase reaction (RT-qPCR) method, and the sensitivity of the ddPCR assay was 10-fold higher. The diagnostic sensitivity and specificity of the ddPCR assay were 100% and did not cross-react with tilapia tissues infected with Tilapia parvovirus, Infectious spleen and kidney necrosis virus, Aeromonas hydrophila, Streptococcus agalactiae, S. iniae and Francisella noatunensis. The assay reproducibility was demonstrated by a high correlation coefficient of 0.998, and the inter-assay coefficients of variability indicated that the ddPCR assay exhibited low variability within and between measurements. The detection limit of the TiLV ddPCR assay was 100 fg cDNA, which is equal to 3.3 copies of TiLV. Furthermore, the ddPCR assay could detect TiLV in mucus, water and infected tissue samples and the lowest copy number of TiLV detected in water samples by the ddPCR assay was 7.9 ± 0.99 copies/reaction The results of the clinical samples tested for TiLV revealed that the ddPCR assay had a relatively higher detection rate than the RT-qPCR method. Overall, the ddPCR method offers a highly promising approach for the absolute quantification of TiLV in carrier fish and samples from the environment with low viral concentrations.

Simultaneous determination of procalcitonin and interleukin-6 in human serum samples with a point-of-care biosensing device

The simultaneous determination of two inflammatory diseases biomarkers, namely procalcitonin (PCT) and interleukin-6 (IL-6), in human serum samples employing a Point-of-Care device based on Multi Area Reflectance Spectroscopy is presented. Dual-analyte detection was achieved using silicon chips with two silicon dioxide areas of different thickness, one functionalized with an antibody specific for PCT and the other with an antibody specific for IL-6. The assay included reaction of immobilized capture antibodies with mixtures of PCT and IL-6 calibrators with the biotinylated detection antibodies, streptavidin and biotinylated-BSA. The reader provided for the automated execution of the assay procedure, as well as for the collection and processing of the reflected light spectrum, the shift of which is correlated to analytes concentration in the sample. The assay was completed in 35 min and the detection limits for PCT and IL-6 were 2.0 and 0.01 ng/mL respectively. The dual-analyte assay was characterized by high reproducibility (the intra- and inter-assay coefficients of variation were less than 10% for both analytes) and accuracy (the percent recovery values ranged from 80 to 113% for both analytes). Moreover, the values determined for the two analytes in human serum samples with the assay developed were in good agreement with the values determined for the same samples by clinical laboratory methods. These results support the potential of the proposed biosensing device application for inflammatory biomarkers determination at the Point-of-Need.

Development of a multiplex qRT-PCR assay for detection of classical swine fever virus, African swine fever virus, and Erysipelothrix rhusiopathiae.

Classical swine fever virus (CSFV), African swine fever virus (ASFV), and Erysipelothrix rhusiopathiae (E. rhusiopathiae) remain endemic in many parts of China. Co-infections make distinguishing their clinical symptoms and pathological changes difficult. This study developed a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) that can simultaneously detect CSFV, ASFV, and E. rhusiopathiae. Three sets of primers and probes were designed to target the CSFV 5΄ untranslated region, ASFV p72 gene, and E. rhusiopathiae 16sRNA gene. Multiplex qRT-PCR for simultaneous differential detection of these three pathogens was developed after optimizing reaction parameters such as annealing temperature, primer and probe concentrations, amplification cycles, etc. The multiplex qRT-PCR could detect CSFV, ASFV, and E. rhusiopathiae simultaneously but could not amplify other porcine pathogens. The assay's limit of detection (LOD) was 2.89 × 102 copies/μL for CSFV, ASFV, and E. rhusiopathiae. All correlation coefficients (R2) at higher than 0.99, and the amplification efficiency was 98, 90, and 84%, respectively. All correlation coefficients (R2) were higher than 0.99, and the efficacy of amplification was 84%. In a repeatability test utilizing standard recombinant plasmids, the intra- and inter-assay coefficients of variation (CVs) were less than 2.27 and 3.79 percent, respectively. Lastly, 150 clinical samples were used to evaluate the assay's applicability in the field. The positive rates of CSFV, ASFV, and E. rhusiopathiae were 1.33%, 0, and 3.33%, respectively. And no co-infection among the three pathogens was found. The concordance rate between the multiplex qRT-PCR and single-plex commercial PCR kits reached 100%. This study's multiplex qRT-PCR could provide a rapid, sensitive, and specific method for the simultaneous and differential detection of CSFV, ASFV, and E. rhusiopathiae.

Development of a Gold Nanoparticle-Linked Immunosorbent Assay of Staphylococcal Enterotoxin B Detection with Extremely High Sensitivity by Determination of Gold Atom Content Using Graphite Furnace Atomic Absorption Spectrometry.

Highly sensitive staphylococcal enterotoxin B (SEB) assay is of great importance for the prevention of toxic diseases caused by SEB. In this study, we present a gold nanoparticle (AuNP)-linked immunosorbent assay (ALISA) for detecting SEB in a sandwich format using a pair of SEB specific monoclonal antibodies (mAbs) performed in microplates. First, the detection mAb was labeled with AuNPs of different particle sizes (15, 40 and 60 nm). Then the sandwich immunosorbent assay for SEB detection was performed routinely in a microplate except for using AuNPs-labeled detection mAb. Next, the AuNPs adsorbed on the microplate were dissolved with aqua regia and the content of gold atoms was determined by graphite furnace atomic absorption spectrometry (GFAAS). Finally, a standard curve was drawn of the gold atomic content against the corresponding SEB concentration. The detection time of ALISA was about 2.5 h. AuNPs at 60 nm showed the highest sensitivity with an actual measured limit of detection (LOD) of 0.125 pg/mL and a dynamic range of 0.125-32 pg/mL. AuNPs at 40 nm had an actual measured LOD of 0.5 pg/mL and a dynamic range of 0.5 to 128 pg/mL. AuNPs at 15 nm had an actual measured LOD of 5 pg/mL, with a dynamic range of 5-1280 pg/mL. With detection mAb labeled with AuNPs at 60 nm, ALISA's intra- and interassay coefficient variations (CV) at three concentrations (2, 8, and 20 pg/mL) were all lower than 12% and the average recovery level was ranged from 92.7% to 95.0%, indicating a high precision and accuracy of the ALISA method. Moreover, the ALISA method could be successfully applied to the detection of various food, environmental, and biological samples. Therefore, the successful establishment of the ALISA method for SEB detection might provide a powerful tool for food hygiene supervision, environmental management, and anti-terrorism procedures and this method might achieve detection and high-throughput analysis automatically in the near future, even though GFAAS testing remains costly at present.

A quadruplex real-time PCR assay combined with a conventional PCR for the differential detection of Marek's disease virus vaccines and field strains.

To evaluate the effect of the vaccine and differentiate vaccine from virulent MDV, a new quadruplex real-time PCR assay based on TaqMan probes was developed to differentiate and accurately quantify HVT, CVI988 and virulent MDV-1. The results showed that the limit of detection (LOD) of the new assay was 10 copies with correlation coefficients >0.994 of CVI988, HVT and virulent MDV DNA molecules without cross-reactivity with other avian disease viruses. The intra-assay and inter-assay coefficients of variation (CVs) of Ct values for the new assay were less than 3%. Analysis of replication kinetics of CVI988 and virulent MDV of collected feathers between 7 and 60 days post-infection (dpi) showed MD5 had no significant effect on the genomic load of CVI988 (p > 0.05), while vaccination with CVI988 could significantly reduce the viral load of MD5 (p < 0.05). Combined with meq gene PCR, this method can effectively identify virulent MDV infections in immunized chickens. These results demonstrated that this assay could distinguish between the vaccine and virulent MDV strains and had the advantages of being reliable, sensitive and specific to confirm the immunization status and monitor the circulation of virulent MDV strains.