Assay For Simultaneous Detection Research Articles

A digital immuno-PCR assay for simultaneous determination of 5-methylcytosine and 5-hydroxymethylcytosine in human serum

This work aimed to develop an ultrasensitive and specific immunosorbent assay for simultaneous detection of double DNA methylation marks. Being considered the most important indicators in disease diagnosis, clinical treatment, and prognosis, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were chosen as the proof-of-concept targets. The described strategy consisted of Phos-tag Biotin anchoring at streptavidin-magnetic nanoparticles, specific immune recognition of anti-5mC antibody and anti-5hmC antibody and labeling of Barcode-antibody, signal amplification of immune PCR and digital PCR machine. Under optimal conditions, the digital immuno-PCR assay showed a board dynamic range from 2.7 × 10−13 mol/L to 2.7 × 10−9 mol/L and the detection limits were 61.7 fmol/L for 5mC, and of 0.111 pmol/L for 5hmC. A 16-fold and 186-fold improvement of LOD were obtained by the proposed approach for 5mC and 5hmC detection compared with real-time immune PCR. The approach also showed ideal specificity, repeatability and stability. The recovery test demonstrated that the digital immuno-PCR assay is a promising platform for the simultaneous determination of the two DNA methylation marks in human serum sample.

Targeted Selected Reaction Monitoring Verifies Histology Specific Peptide Signatures in Epithelial Ovarian Cancer.

Simple SummaryOvarian cancer is a lethal disease due to its late phase discovery. Any steps towards improving early diagnostics will dramatically increase survival rates. To identify new ovarian cancer biomarker panels, we need to focus on early-stage disease and all histologic subtypes. In this study we have, based on prior discoveries, constructed a multiplexed targeted selected-reaction-monitoring assay to detect peptides from 177 proteins in only 20 µL of plasma. The assay was evaluated in patients with a focus on early-stages and all ovarian cancer histologies in separate groups. With multivariate analysis, we found the highest predictive value in the benign vs. low-grade serous (Q2 = 0.615) and mucinous (Q2 = 0.611) early stage compared to all malignant (Q2 = 0.226) or late stage (Q2 = 0.43) ovarian cancers. The results show that each ovarian cancer histology subgroup can be identified by a unique panel of proteins.Epithelial ovarian cancer (OC) is a disease with high mortality due to vague early clinical symptoms. Benign ovarian cysts are common and accurate diagnosis remains a challenge because of the molecular heterogeneity of OC. We set out to investigate whether the disease diversity seen in ovarian cyst fluids and tumor tissue could be detected in plasma. Using existing mass spectrometry (MS)-based proteomics data, we constructed a selected reaction monitoring (SRM) assay targeting peptides from 177 cancer-related and classical proteins associated with OC. Plasma from benign, borderline, and malignant ovarian tumors were used to verify expression (n = 74). Unsupervised and supervised multivariate analyses were used for comparisons. The peptide signatures revealed by the supervised multivariate analysis contained 55 to 77 peptides each. The predictive (Q2) values were higher for benign vs. low-grade serous Q2 = 0.615, mucinous Q2 = 0.611, endometrioid Q2 = 0.428 and high-grade serous Q2 = 0.375 (stage I–II Q2 = 0.515; stage III Q2 = 0.43) OC compared to benign vs. all malignant Q2 = 0.226. With targeted SRM MS we constructed a multiplexed assay for simultaneous detection and relative quantification of 185 peptides from 177 proteins in only 20 µL of plasma. With the approach of histology-specific peptide patterns, derived from pre-selected proteins, we may be able to detect not only high-grade serous OC but also the less common OC subtypes.

Detection of PAX5 Overexpression By RNA-Seq for B-Cell Lineage Detection in Acute Lymphocytic Leukemia

Background: Paired-box Pax gene family protein 5 (PAX5)/B-cell specific activator protein (BSAP) is a transcription factor encoded by the PAX5 gene and has an essential role in B-cell differentiation and maturation. High PAX5 expression is detected ensures commitment to B-cell lineage. PAX5 is normally downregulated at the plasma cell stage of B-cell development. Complete or partial deletion of the PAX5 gene has been found as secondary event associated with BCR-ABL1 or TCF3-PBX1 fusions in Acute Lymphocytic Leukemia (ALL) cases. PAX5 expression is a diagnostic marker for B-cell lineage and may help quantify minimal residual disease in B-ALL. Lineage determination of leukemic blasts is most often performed by flow cytometry, but also by immunohistochemistry (IHC). Evaluation of PAX5 is most commonly available by IHC and is not widely performed by flow cytometry. In cases with limited specimen for evaluation or aberrant loss of some B-cell markers, determining quantitative levels of RNA from lineage-specific genes, such as PAX5, could be a valuable clinical diagnostic tool for ALL patients.Our existing single tube NGS based assay for simultaneous detection of DNA alterations and RNA fusions in heme malignancies from Total Nucleic Acid (TNA), can also be used to detect PAX5 gene expression through select exons enrichment along with a total of 213 genes. However, one of the current challenges for NGS-based gene expression profiling is to setup a threshold for overexpression. Here we developed a cutoff criterion for PAX5 overexpression and evaluated the performance of PAX5 gene expression analysis using the in-use heme assay and its potential use in clinical laboratory for cell lineage detection.Methods: RNA sequencing was performed on TNA extracted from ALL samples and from 32 healthy donors using partial anchored amplicon based (Qiagen, inc) heme NGS assay. PAX5 RNA expression was calculated by TPM (transcript per million) counts normalized to TPM of the house-keeping gene GUSB. A commercially available qRT-PCR assay was used as orthogonal method to confirm the gene expression. The expression call by NGS based on the normalized value was confirmed by a commercial qRT-PCR assay in house validated through serial dilutions of template for six log scale. The analytical cutoff was determined from normalized TPM calculation from 32 healthy volunteers following CLSI guideline (CLSI_EP17-A2) and evaluated the outcome with IHC positive /negative clinical samples (a CLIA validated assay). Further, we used the established cutoff to evaluate the sensitivity or specificity in cohort of ALL samples.Results: In this study we established the cutoff for PAX5 gene over-expression using the currently in-use heme NGS assay. First, a cutoff was established following the method in the CLSI guidelines and tested for sensitivity and specificity in the ALL sample cohort. PAX5 TPM normalization to GUSB or to the geometric mean of four house keeping genes (GUSB, PGD, RPL5 and RPL19) showed a strong correlation (R2>0.95), and GUSB was selected for further normalization since GUSB TPM values were most conserved across all the samples. Independent in-house evaluation for commercial qRT-PCR assay showed efficiency at 94.3 and 96% for GUSB and PAX5, respectively (with linearity R2>0.95), and been used to compare the NGS and IHC data as independent orthogonal assay. When a cohort of samples for Pax5 by IHC (positive and negative), a sensitivity at 67% and specificity at 100% were observed for the NGS based Pax5 detection. NGS results on the discordant samples were confirmed by qRT-PCR to have low RNA expression. Notably the discordant, IHC positive samples contained very low numbers of B cells. Alongside with other possible mechanisms of increased protein levels such as increased protein translation/increased protein stability could explain the discordance between RNA expression and the protein detection by IHC.Conclusions: In this study we demonstrate that NeoGenomics's (heme) NGS assay can be used for PAX5 gene over-expression analysis on ALL. The heme NGS is inexpensive and is already integrated in the benchwork workflow without adding extra burden and can be used as an objective quantification of PAX5 levels overcoming the challenges associated with the relative signal intensity biases in IHC testing. This type of RNA testing can be useful especially with specimens having limited material. DisclosuresGhosal: NeoGenomics Laboratories: Current Employment. Alarcon: NeoGenomics Laboratories: Current Employment. Koo: Neo Genomics Laboratories: Current Employment. Kang: Neo Genomics Laboratories: Current Employment. Ramesh: Neo Genomics Laboratories: Current Employment. Gyuris: Neo Genomics Laboratories: Current Employment. Jung: NeoGenomics Laboratories, Inc.: Current Employment. Thomas: NeoGenomics Laboratories, Inc.: Current Employment. Fabunan: NeoGenomics Laboratories, Inc.: Current Employment. Magnan: NeoGenomics Laboratories, Inc.: Current Employment. Nam: NeoGenomics Laboratories, Inc.: Current Employment. Petersen: Neo Genomics Laboratories: Current Employment. Lopez-Diaz: NeoGenomics Laboratories, Inc.: Current Employment. Bender: NeoGenomics Laboratories, Inc.: Current Employment. Agersborg: NeoGenomics Laboratories, Inc.: Current Employment. Ye: Neo Genomics Laboratories: Current Employment. Funari: NeoGenomics Laboratories, Inc.: Current Employment.

A universal ELISA assay for detecting six strains of ovine Babesia species in China

Ovine babesiosis, caused by genus of Babesia, is a zoonotic disease and mainly transmitted by hard ticks. It has led to enormous economic losses to the sheep industry in China. In the present study, an ELISA assay for simultaneous detection six strains of Babesia spp., including B. motasi Lintan, B. motasi Tianzhu, B. motasi Hebei, B. motasi Ningxian, Babesia sp. Xinjiang and Babesia sp. Dunhuang, was developed using Apical Membrane Antigen 1 (AMA1) as candidate diagnostic antigen. The sensitivity and specificity of the established ELISA were 97.4 % and 98.0 %, respectively. Relatively high level of specific antibodies could be detected from 12th day to 126th day after sheep experimentally infected with Babesia spp.. A small scale of field sera was investigated using the developed ELISA assay, and the average positive rate was 51.98 %. This study provides an easy to operate, cost effective and time saving approach, which is suitable for both field and experimental samples, thus it could be a useful tool in epidemiological investigations and diagnoses of ovine babesiosis.

A homogeneous nucleic acid assay for simultaneous detection of SARS-CoV-2 and influenza A (H3N2) by single-particle inductively coupled plasma mass spectrometry

In recent years, single particle inductively coupled plasma mass spectrometry (SP-ICP-MS) has become a powerful tool for biological quantitative analysis. Homogeneous analysis method requires no separation and washing steps, which is suited for the analysis of highly infectious pathogens, so as to reduce the risk of infection during the operation. SARS-CoV-2 spreads all over the world, and its early infection symptoms are similar to influenza, which brings inconvenience to triage. Therefore, developing novel analytical method for simultaneous detection of multiple viral nucleic acids is essential. Taking the advantages of SP-ICP-MS and homogeneous analysis strategy, a SP-ICP-MS homogeneous nucleic acid assay by using gold nanoparticles (Au NPs) and silver nanoparticles (Ag NPs) probes was established for simultaneous sensitive analysis of SARS-CoV-2 and influenza A (H3N2). In the present of target SARS-CoV-2 or H3N2 nucleic acids, corresponding Au NPs or Ag NPs probes form larger aggregates, resulting in increased pulse signal intensity and reduced pulse signal frequency of the corresponding NPs in SP-ICP-MS measurement. In this assay, the reaction system of Au NPs and Ag NPs probes does not interfere with each other, and there was no separation and washing procedure, which facilitates operation, saves the analysis time, and improves the analysis efficiency. The linear range of this method is 5–1000 pmol L−1, with low-level limits of quantification of target nucleic acid. The developed SP-ICP-MS simultaneous homogeneous detection method has a good potential for detecting nucleic acid, protein, cell and other biological samples by changing different modification sequences on the NPs probes.

Rapid Detection and Differentiating of the Predominant Salmonella Serovars in Chicken Farm by TaqMan Multiplex Real-Time PCR Assay.

Salmonella has been known as an important zoonotic pathogen that can cause a variety of diseases in both animals and humans. Poultry are the main reservoir for the Salmonella serovars Salmonella Pullorum (S. Pullorum), Salmonella Gallinarum (S. Gallinarum), Salmonella Enteritidis (S. Enteritidis), and Salmonella Typhimurium (S. Typhimurium). The conventional serotyping methods for differentiating Salmonella serovars are complicated, time-consuming, laborious, and expensive; therefore, rapid and accurate molecular diagnostic methods are needed for effective detection and prevention of contamination. This study developed and evaluated a TaqMan multiplex real-time PCR assay for simultaneous detection and differentiation of the S. Pullorum, S. Gallinarum, S. Enteritidis, and S. Typhimurium. In results, the optimized multiplex real-time PCR assay was highly specific and reliable for all four target genes. The analytical sensitivity corresponded to three colony-forming units (CFUs) for these four Salmonella serovars, respectively. The detection limit for the multiplex real-time PCR assay in artificially contaminated samples was 500 CFU/g without enrichment, while 10 CFU/g after pre-enrichment. Moreover, the multiplex real-time PCR was applied to the poultry clinical samples, which achieved comparable results to the traditional bacteriological examination. Taken together, these results indicated that the optimized TaqMan multiplex real-time PCR assay will be a promising tool for clinical diagnostics and epidemiologic study of Salmonella in chicken farm and poultry products.

Comparison of Alinity m HPV and cobas HPV assays on cervical specimens in diverse storage media

ObjectiveTo assess the concordance of high-risk HPV (HR-HPV) testing with the Alinity assay on cervical samples collected with diverse collection/storage protocols (ThinPrep, SurePath, Cervicollect) and to assess inter-assay concordance of HR-HPV testing of cervical cell specimens with Alinity m HR HPV assay (Alinity) vs cobas® 4800 HPV assay (cobas). MethodsSpecimens were obtained from 560 women attending a Women's Health clinic. Two specimens were obtained from each woman with combinations of two of the three collection devices and aliquots were tested by the two assays. ResultsAlinity showed an agreement of 93.9%, Kappa = 0.89 (263/280) between ThinPrep and SurePath specimens; 97.5%, Kappa = 0.95 (347/356) and 92.9%, Kappa = 0.85 (104/112) between ThinPrep and SurePath aliquots taken before or after cytology processing, respectively. Cervi-Collect specimens showed an agreement of 94.6%, Kappa = 0.89 (265/280) with ThinPrep specimens. Compared to cobas, Alinity showed agreements of 94.3%, Kappa = 0.88 (395/419) and 91.8%, Kappa = 0.82 (257/280) between ThinPrep and SurePath specimens, respectively. Alinity and cobas detected genotypes 16/18 and other high-risk HPV types at similar rates and showed similar correlations with cytology grades. ConclusionsCompared to cobas, Alinity performed equally well for detecting HPV in cervical specimens obtained with ThinPrep and SurePath. The Cervi-Collect device compared well to the other collection methods. Alinity is a reliable assay for simultaneous detection of HPV-16/18 and other high-risk genotypes in cervical specimens.

Dual quantum dot nanobeads-based fluorescence-linked immunosorbent assay for simultaneous detection of aflatoxin B1 and zearalenone in feedstuffs

A novel dual quantum dot nanobeads-based fluorescence-linked immunosorbent assay (QBs-FLISA) was successfully developed for simultaneously detecting aflatoxin B1 (AFB1) and zearalenone (ZEN) in feedstuffs. Dual CdSe/ZnS quantum dot nanobeads with different diameters that emit red and green fluorescence were conjugated with anti-AFB1 and anti-ZEN monoclonal antibodies to prepare fluorescent probes, which greatly enhance analytical performance. Under the optimal conditions, the limits of detection for AFB1 and ZEN were 9.3 and 102.1 pg mL−1, respectively. The recoveries ranged from 82.50% to 116.21% with relative standard deviation less than 11.3%. Compared with traditional enzyme-linked immunosorbent assay, detection sensitivities of AFB1 and ZEN using QBs-FLISA were increased 20 and 5 folds, respectively. In addition, results of feedstuff samples analyzed by QBs-FLISA and liquid chromatography tandem mass spectrometry showed a good agreement (R2 = 0.99).

A metabarcoding approach for detecting protozoan pathogens in wild oysters from Prince Edward Island, Canada

Food and waterborne protozoan pathogens including Cryptosporidium parvum, Giardia enterica and Toxoplasma gondii are a global concern for human public health. While all three pathogens have been detected in commercial shellfish, there is currently no standard approach for detecting protozoan parasites in shellfish. Common molecular and microscopic methods are limited in the number of pathogens they can simultaneously detect and are often targeted at one or two of these pathogens. Previously, we developed and validated a novel 18S amplicon-based next-generation sequencing assay for simultaneous detection of Cryptosporidium spp., Giardia spp. and T. gondii in shellfish. In this study, we applied the assay for protozoan pathogen detection in wild oysters from Prince Edward Island (PEI). Oysters were harvested from restricted and prohibited areas, classified by the Canadian government according to fecal coliform counts in surrounding waters, and different fractions (whole tissue homogenate and hemolymph) were analyzed. Protozoan DNA was detected using metabarcoding in 28%, of oysters tested (N = 128), and the pathogen read counts in oyster homogenate were considerably higher than those in hemolymph. Protozoan read count thresholds were established for classifying probable oyster contamination with pathogens to account for low levels of background protozoan reads detected in negative controls. Assay results showed protozoan contamination was not associated with harvesting site classifications, suggesting that using fecal indicators for ensuring food safety may be insufficient. Due to the complex matrix, an oyster DNA reduction step may further improve the pathogen detection sensitivity of the assay. Results from this study affirm that novel metabarcoding is a promising screening tool for detection of protozoan pathogens in shellfish.

Fluorescent hollow ZrO2@CdTe nanoparticles-based lateral flow assay for simultaneous detection of C-reactive protein and troponin T.

Highly fluorescent hollow ZrO2@CdTe nanoparticles (NPs) were synthesized efficiently via thehydrothermal method. By changing the hydrothermal time of ZrO2@CdTe NP, the peaks of fluorescence spectra measured at fluorescent excitation of 330nm were at540nm, 590nm, and 640nm, respectively. Hollow ZrO2 NPs have a uniform core-shell structure with the size of 178 ± 10nm and shell of 19 ± 4nm. The as-prepared yellow-ZrO2@CdTe NPs were used to develop lateral flow assay (LFA) for the sensitive and qualitative detection of C-reactive protein (CRP). The visual limit of detection of the LFA for the CRP antigen was 1 μg/L within 20min, which is1000-fold lower than that ofcolloidal gold-based LFA. In addition, a multiplex lateral flow assay (mLFA) was developed using the as-prepared green and red-ZrO2@CdTe NPs for the simultaneous, specific, sensitive, and qualitative detection of CRP and troponin T (cTnT). The visual limits of detection of CRP and cTnT in mLFA were 10μg/L and 0.1mg/L, respectively. The excellent performance of ZrO2@CdTe NPs should facilitate their application in point-of-care technology for the detection of other biomarkers.

Development of multiplex real-time PCR assays for differential detection of capripoxvirus, parapoxvirus and foot-and-mouth disease virus.

This study reports the development of multiplex real-time PCR assays for differential detection of capripoxvirus (CaPV), parapoxvirus (PaPV) and foot-and-mouth disease virus (FMDV) in sheep, goats and cattle. Three multiplex assays were developed, a capripox (CaP) rule-out assay for simultaneous detection and differentiation of CaPV and PaPV, a FMD rule-out assay for simultaneous detection and differentiation of FMDV and PaPV, and a FMD/CaP rule-out assay for simultaneous detection and differentiation of CaPV, PaPV and FMDV. All multiplex assays included β-actin gene ACTB as an internal positive control to monitor PCR inhibition and accuracy of nucleic acid extractions. The optimized assays were highly specific to the target viruses (CaPV, PaPV and FMDV) with no cross-reactivity against other viruses that cause similar clinical signs. Using positive control plasmids as template, the limit of detection (LOD) of the multiplex assays were estimated as 2 CaPV, 7 PaPV and 15 FMDV copies per assay. The amplification efficiency (AE) and correlation coefficient (R2 ), estimated from the standard curves (Ct vs. log10 template dilution), were 94%-106% and >0.99, respectively, for CaP and FMD rule-out assays, 96%-116% (AE) and >0.98 (R2 ), respectively, for CaP/FMD rule-out assays and 91%-102% and >0.99, respectively, for the corresponding singleplex assays. The diagnostic sensitivity (DSe) of the multiplex assays was assessed on 35 CaPV and 39 FMDV clinical specimens from experimentally infected (CS-E) animals, and 29 CaPV (LSDV), 28 FMDV and 36 PaPV clinical specimens from naturally infected (CS-N) animals; all tested positive (DSe 100%) except two CS-E FMDV specimens that were tested negative by FMD rule-out and the corresponding singleplex (FMDV) assays (37/39; DSe 95%). The newly developed multiplex assays offer a valuable tool for differential detection of clinically indistinguishable CaPV, PaPV and FMDV in suspected animals and animals with mixed infections.

A Multiplex Assay for Simultaneous Detection of Three Retroviruses in Nonhuman Primates Based on the Luminex xTAG Platform

A Multiplex Assay for Simultaneous Detection of Three Retroviruses in Nonhuman Primates Based on the Luminex xTAG Platform

Multiplex detection of "Candidatus Liberibacter asiaticus" and Spiroplasma citri by qPCR and droplet digital PCR.

"Candidatus Liberibacter asiaticus" (CLas) and Spiroplasma citri are phloem-limited bacteria that infect citrus and are transmitted by insect vectors. S. citri causes citrus stubborn disease (CSD) and is vectored by the beet leafhopper in California. CLas is associated with the devastating citrus disease, Huanglongbing (HLB), and is vectored by the Asian citrus psyllid. CLas is a regulatory pathogen spreading in citrus on residential properties in southern California and is an imminent threat to spread to commercial citrus plantings. CSD is endemic in California and has symptoms in citrus that can be easily confused with HLB. Therefore, the objective of this study was to develop a multiplex qPCR and duplex droplet digital PCR (ddPCR) assay for simultaneous detection of CLas and S. citri to be used where both pathogens can co-exist. The multiplex qPCR assay was designed to detect multicopy genes of CLas-RNR (5 copies) and S. citri-SPV1 ORF1 (13 copies), respectively, and citrus cytochrome oxidase (COX) as internal positive control. Absolute quantitation of these pathogens was achieved by duplex ddPCR as a supplement for marginal qPCR results. Duplex ddPCR allowed higher sensitivity than qPCR for detection of CLas and S. citri. ddPCR showed higher tolerance to inhibitors and yielded highly reproducible results. The multiplex qPCR assay has the benefit of testing both pathogens at reduced cost and can serve to augment the official regulatory protocol for CLas detection in California. Moreover, the ddPCR provided unambiguous absolute detection of CLas and S. citri at very low concentrations without any standards for pathogen titer.

Recent advances in nanotechnology for simultaneous detection of multiple pathogenic bacteria

Owing to the environmental complexity in terms of the coexistence of multiple pathogenic microorganisms, development of the simple, rapid, and accurate assay for simultaneous detection of multiple pathogenic microorganisms still faces major challenges, but it is an unavoidable topic. Recently, with the thriving development of nanotechnologies, nanoparticle-based assays are emerging as one of the most promising methods to enable the accurate identifying and quantifying of multiple pathogenic microorganisms with the merits of cost-efficiency, time-saving, high sensitivity and selectivity. Regarding to the efforts that have been made in this field, in this review, we have systematically summarized the recent progress in the development of functionalized-nanoparticles for the simultaneous detection of multiple pathogenic bacteria. In detail, five different types of nanomaterials, including magnetic nanoparticles, gold nanomaterials, inorganic quantum dots (QDs), upconversion nanoparticles (UCNPs), and carbon nanomaterials in the simultaneous detection of multiple pathogenic bacteria are reviewed, and the confronting challenges as well as a brief outlook are also discussed.

Evaluation of a measles virus multiplex, triple-target real-time RT-PCR in three specimen matrices at a U.S. academic medical center.

Measles virus (MeV) is an important cause of acute febrile illness and pediatric mortality globally, with recent U.S. outbreaks associated with under-vaccination. MeV is highly contagious and timely diagnosis is critical to limit spread. RNA detection is the most sensitive method for acute measles diagnosis; however, MeV nucleic acid amplification assays are not widely available. We performed a diagnostic accuracy study of a triple-target, real-time RT-PCR (rRT-PCR) assay for simultaneous detection of MeV N, H, and L genes. The MeV triple-target rRT-PCR was tested against serial dilutions (7.0-2.0 log10 copies/mL) of five MeV isolates representing circulating genotypes, and detected 98.7% (74/75) of nasopharyngeal (NP) swab dilutions, 100% (75/75) of plasma dilutions, and 85.3% (64/75) of urine dilutions. MeV RNA detection in urine was markedly improved with the addition of a nucleic acid stabilizing agent. A 95% lower limit of detection (LLOD) of < 3.0 log10 copies/mL was established in each specimen matrix. No cross-reactivity with relevant viruses or interfering substances were identified in specificity studies. The MeV triple-target rRT-PCR detected all three gene targets in a clinical NP swab from an individual with confirmed measles infection. Furthermore, pooled testing from 798 influenza A/B/RSV-negative pediatric NP swabs identified two specimens positive for MeV RNA, confirmed by N gene sequencing to represent shedding of the vaccine-type measles virus. The MeV triple-target rRT-PCR assay showed high analytic sensitivity across circulating MeV genotypes in three clinically-relevant matrices. Implementation of this assay in the clinical laboratory may facilitate timely diagnosis of acute measles infection and implementation of appropriate infection control interventions.

Establishment of Duplex SYBR Green I-based Real-time PCR Assay for Simultaneous Detection of Duck Hepatitis A Virus-1 and Duck Astrovirus-3.

Duck viral hepatitis (DVH) mainly affects ducklings under 1 month of age, causes liver necrosis, enlargement, and hemorrhage, and is highly lethal, seriously jeopardizing the duck industry. The prevalence of duck hepatitis A virus (DHAV-1) and duck astrovirus type 3 (DAstV-3) is increasing, and coinfection is common. Moreover, the similar clinical characteristics of the DHAV-1 and DAstV-3 infections and the high frequency of coinfection make diagnosis difficult. In this study, to establish a method for the rapid, simultaneous detection of DHAV-1 and DAstV-3, two pairs of specific primers were designed according to their conserved gene regions. An SYBR® Green I-based qPCR assay was successfully established that can quickly and differentially detect the two viruses. Moreover, the assay is highly specific and does not show cross-reaction with other common viruses. The detection limit of the method is 7.34 × 101 copies/µl and 3.78 × 101 copies/µl for DHAV-1 and DAstV-3, respectively, indicating high sensitivity. A total of 34 clinical samples were tested using the established method; the positive rates for DHAV-1 and DAstV-3 were 14.71% and 8.82%, respectively, and that for coinfection was 2.94% (1/34), which was better than that obtained with conventional PCR. In summary, the SYBR Green I-based qPCR assay established in this study has high specificity, good sensitivity and accuracy, high feasibility, and is rapid. Thus, it can be a powerful tool for the coinfection detection of DHAV-1 and DAstV-3 and for future epidemiologic studies.

A novel α-Fe2O3 nanocubes-based multiplex immunochromatographic assay for simultaneous detection of deoxynivalenol and aflatoxin B1 in food samples

Multiple mycotoxins may co-occurred in a single product and cause serious threats to public health. Herein, a multiplex immunochromatographic assay (mICA) was developed based on novel α-Fe2O3 nanocubes (FNCs) as signal tags for the simultaneous detection of deoxynivalenol (DON) and aflatoxins B1 (AFB1). The FNCs with eminent water-dispersibility and stability were synthesized by a one-step hydrothermal process, which can rapidly label monoclonal antibodies (mAbs) to form FNCs-mAb probe. Under optimal experimental conditions, the visual limit of detection for DON and AFB1 were 0.18 ng/mL and 0.01 ng/mL, respectively, which were at least 2-fold improvement compared with the traditional gold nanoparticles based mICA. Furthermore, DON and AFB1 residual in mung bean, millet and corn were successfully detected by the proposed biosensor, and the average recovery rates were 93.3%–128.3%. Thus, this work indicated that the developed FNCs-mICA provided a sensitive, rapid and reliable on-site simultaneous detection of multiple mycotoxins.

Development of an ultrasensitive multiplex assay for simultaneous detection of Aβ1‐42, Aβ1‐40, GFAP and NF‐L in blood

AbstractBackgroundPrevious work (1) has described plasma amyloid, GFAP and NF‐L as predictors of Alzheimer’s pathology. Here we build on the Amyblood 1‐42/1‐40 Simoa assay (1,2) where GFAP and NF‐L, as indicators of astrogliosis and neurodegeneration, further added to the prediction for Aβ PET imaging or diagnosis. We report a Simoa multiplex assay that simultaneously measures Aβ1‐42, Aβ1‐40, GFAP and NF‐L in blood.MethodsA 4‐plex immunoassay was designed using Simoa technology. A monoclonal capture antibody specific for each analyte was attached to one of four different fluorescent dye‐encoded beads. Antibodies against Aβ1‐42 and Aβ1‐40 were from ADx (clones 21F12 and 2G3 as capture; 3D6 as detector), NF‐L and GFAP antibodies were as supplied in Quanterix commercial kits. The assay was further evaluated for its analytical performance, including analyte detectability in commercially‐sourced human plasma (n=20) and CSF (n=10) from healthy controls, and for discrimination between AD patient and control plasma (each n=10) from the Alzheimer Center Amsterdam.ResultsAssay calibration ranges were 0.041‐30pg/mL (Aβ1‐42), 0.016‐12pg/mL (Aβ1‐40), 6.86‐5000pg/mL (GFAP), and 0.686‐500pg/mL (NF‐L). Lower Limits of detection were 0.148pg/mL (Aβ1‐42), 0.084pg/mL (Aβ1‐40), 1.66pg/mL (GFAP), 0.157pg/mL (NF‐light). Limits of quantification for assay prototype were 0.381pg/mL (Aβ1‐42), 0.125pg/mL (Aβ1‐40), 4.99pg/mL (GFAP), 0.493pg/mL (NF‐light). In parallelism studies, mean recovery of serially diluted endogenous plasma and CSF (each n=4) was within 80‐120%. Grand mean recovery of expected spike concentration was within 70‐130% (n=4). The assay has 100% detectability for all four analytes in both plasma and CSF samples. NF‐L and GFAP concentrations increased in AD samples (respectively p&lt;0.05 and 0.01) while the Aβ 42/40 ratio decreased (p&lt;0.05) in AD samples versus healthy donors.ConclusionsThis multiplex Simoa assay accurately and specifically measure four biomarkers simultaneously in plasma and CSF, providing a new tool to quantify Aβ1‐42, Aβ1‐40, GFAP and NF‐L. Ongoing studies using a larger cohort of clinical samples will determine the potential clinical utility of this assay.

Epidemiological and viral features of a cohort of SARS-CoV-2 symptomatic and asymptomatic individuals in an area of the Colombian Caribbean

BackgroundSevere acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is an emerging viral pandemic disease. In the last 6 months, SARS-CoV-2 has caused millions of reported cases and hundreds of thousands of deaths. As other world regions, South America has not contained the pandemic’s advance since it lacks the hospital and economic capacities. Public health implications of transmission, while the asymptomatic/presymptomatic infection is a critical concern at the current pandemic.ObjectiveDescribe the socio-demographic, clinical, and viral features of a cohort of SARS-CoV-2 infected individuals from the Colombian Caribbean.MethodsSix hundred eighty-six clinical samples of suspected SARS-CoV-2 infection cases and contacts individuals from several hospital centers in the department of Córdoba, Colombia, were received at our laboratory between April 9th and May 16th, 2020. RNA was extracted using lysis buffers and spin columns. The samples were tested for SARS-CoV-2 by reverse transcription real-time polymerase chain reaction (RT-qPCR) using commercially available multiplex real-time PCR assay for simultaneous detection of 3 target genes of SARS-CoV-2 (Allplex™, 2019-nCoV assay, Korea). Viral copies quantification was done using a standard curve constructed from seriated dilutions of a SARS-CoV-2 positive control. Statics descriptive methods were used.ResultsThirty-five nasopharyngeal samples were positive for SARS-CoV-2 infection; the average age was 43 (range, 1–95 years). Seventeen of 35 (49%) of the patients showed symptoms. Most of them had a cough, fever, and odynophagia; three of the patients reported having arthralgia. Only two patients required hospitalization. None of the patients had known co-morbidities. RT-qPCR results show that two of the symptomatic patients had significantly higher RNA copies than the rest. Eighteen of 35 (51%) individuals were asymptomatic, and the average age was 30 (range, 6–61 years). Four asymptomatic individuals showed a higher copy than some symptomatic patients; nonetheless, the average of RNA copies 8.26 × 1010 was lower than the symptomatic.ConclusionsThis study shows that asymptomatic patients may develop infections with a high number of RNA copies. Since a considerable percentage of infections may be asymptomatic/presymptomatic, enhanced testing approaches may be needed to detect these persons. Due the occurrence of a large proportion of infections being a result from transmission originated in asymptomatic/presymptomatic individuals, public health interventions in Colombia should be based on two steps: a massive molecular screening, and viral load quantification. Finally, a remarkable issue in our study is the average age of symptomatic and asymptomatic groups (43 and 30 respectively) which may be important because of the economic impact that has been caused by the coronavirus pandemic and may be probably the cause of the reduced lethality observed in the country and the department at the time of this study.

Development a multiplex RT-PCR assay for simultaneous detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus

African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused considerable financial losses to the pig industry worldwide, and it is critical to achieve early and accurate diagnosis of these viruses to control the diseases induced by them. In this study, three pairs of specific primers were designed based on the highly conserved genome regions of these viruses, and a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay for ASFV, CSFV and APPV was established after various reaction conditions were optimized. The mRT-PCR assay consisted of two steps, that is, reverse transcription (RT) and mPCR. The assay was highly specific, sensitive, and reproducible for ASFV, CSFV and APPV without cross-reaction with other swine pathogens. The sensitivity of this assay, which used purified plasmid constructs containing specific viral target fragments as templates, was 6.34 × 102 copies/μL for ASFV and 6.34 × 101 copies/μL for both CSFV and APPV. A total of 384 clinical samples from piglets suspected to be infected in Guangxi Province, Southern China, during 2018−2019 were analyzed by the established mRT-PCR method. The results showed that the positive rates of ASFV, CSFV and APPV were 43.75 %, 13.28 % and 4.17 %, respectively, and the coinfection rates of ASFV/CSFV, ASFV/APPV and CSFV/APPV were 5.47 %, 1.83 % and 1.30 %, respectively. To understand the epidemiological characteristics of APPV, the newly discovered virus, in Guangxi Province, the clinical samples from APPV-positive animals were selected randomly for amplification and sequencing, and the complete genomic sequences of four APPV strains were obtained. Phylogenetic analysis demonstrated that APPV strains from Guangxi Province had a high degree of genetic diversity. This study provides an important tool for rapid detection and accurate diagnosis of ASFV, CSFV and APPV.