Detection Limit Of Assay Research Articles

Charge Transfer Complex of Lorlatinib with Chloranilic Acid: Characterization and Application to the Development of a Novel 96-Microwell Spectrophotometric Assay with High Throughput.

Lorlatinib (LRL) is the first drug of the third generation of anaplastic lymphoma kinase (ALK) inhibitors used a first-line treatment of non-small cell lung cancer (NSCLC). This study describes, for the first time, the investigations for the formation of a charge transfer complex (CTC) between LRL, as electron donor, with chloranilic acid (CLA), as a π-electron acceptor. The CTC was characterized by ultraviolet (UV)-visible spectrophotometry and computational calculations. The UV-visible spectrophotometry ascertained the formation of the CTC in methanol via formation of a new broad absorption band with maximum absorption peak (λmax) at 530 nm. The molar absorptivity (ε) of the complex was 0.55 × 103 L mol-1 cm-1 and its band gap energy was 2.3465 eV. The stoichiometric ratio of LRL/CLA was found to be 1:2. The association constant of the complex was 0.40 × 103 L mol-1, and its standard free energy was -0.15 × 102 J mole-1. The computational calculation for the atomic charges of an energy minimized LRL molecule was conducted, the sites of interaction on the LRL molecule were assigned, and the mechanism of the reaction was postulated. The reaction was adopted as a basis for developing a novel 96-microwell spectrophotometric method (MW-SPA) for LRL. The assay limits of detection and quantitation were 2.1 and 6.5 µg/well, respectively. The assay was validated, and all validation parameters were acceptable. The assay was implemented successfully with great precision and accuracy to the determination of LRL in its bulk form and pharmaceutical formulation (tablets). This assay is simple, economic, and more importantly has a high-throughput property. Therefore, the assay can be valuable for routine in quality control laboratories for analysis of LRL's bulk form and pharmaceutical tablets.

Abstract 2109: Analytical characterization of a multiplex panel measuring 45 cytokines by proximity extension assay in plasma from subjects with NSCLC

Abstract Introduction Cytokines are fundamental regulators of immune system processes and are responsible for the differentiation, proliferation, and function of immune cells. With many anti-cancer therapeutic strategies focused on targeting or harnessing the immune system, or components thereof, quantification of circulating cytokines has become important to describe the pharmacodynamic and mechanistic effects of these investigational therapies. To support these efforts, new multiplexed immunoassay panels have been developed to expand the available options for broad spectrum characterization of cytokine profiles from biospecimens. Here we characterize the analytical performance of one such panel based on proximity extension assay technology to measure 45 cytokines in plasma. Experimental Procedures Plasma samples (n = 27) from patients with non-small cell lung cancer (NSCLC), along with age-matched plasma samples (n = 6) from healthy normal subjects were analyzed using the Olink Cytokine 48 kit. Samples were thawed and processed according to manufacturer instructions for the incubation, extension, and detection on the Olink Signature Q100 instrument. A subset of the screened samples was run in quadruplicate across multiple runs and analyzed on two separate instruments. Results Of the 45 cytokines assayed, 41 had values above the assay limit of detection in ≥75% of samples analyzed. When compared to the normal human plasma, 26 cytokines were significantly elevated (p-value ≤ 0.05) in plasma from NSCLC subjects, including those known to be associated both with poor (e.g., IL-1β, IL-6) and good prognosis (e.g., IFN-γ). Furthermore, many of the cytokines with significant differences were of low abundance; 13 of the 26 cytokines had mean values ≤ 20.0 pg/mL. For two of the incurred samples which were run across multiple replicates and runs, with mid- to high- levels of cytokines, the median intra-assay CV across all assays was 6.2% with 90% of the assays demonstrating CVs of less than 12%. This only increased to a 7.7% median CV in inter-run comparison with 90% of the assays remaining below 12%. Additional results from the incurred sample analysis will be presented. Conclusions These results demonstrate that multiplex cytokine analysis, based on proximity extension assays, can be effectively deployed to screen a large number of targets and can uncover clinically relevant changes in low abundant cytokines. Citation Format: Anne Jang, Daniel Feingold, Mark H. Watson, Gwenaël Pottiez, Rudolf Guilbaud, Nicholas Francois Dupuis. Analytical characterization of a multiplex panel measuring 45 cytokines by proximity extension assay in plasma from subjects with NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2109.

Sensitive detection of viable salmonella bacteria based on tertiary cascade signal amplification via splintR ligase ligation-PCR amplification-CRISPR/Cas12a cleavage

Several viable Salmonella bacteria are capable of causing severe human diseases and huge economic losses. In this regard, viable Salmonella bacteria detection techniques that can identify small numbers of microbial cells are highly valuable. Here, we present a detection method (referred to as SPC) based on the amplification of tertiary signals using splintR ligase ligation, PCR amplification and CRISPR/Cas12a cleavage. The detection limit of the SPC assay was 6 copies (HilA RNA) and 10CFU (cell). Based on Intracellular HilA RNA detection, this assay can be used to distinguish between viable and dead Salmonella. In addition, it is able to detect multiple serotypes of Salmonella and has been successfully used to detect Salmonella in milk or isolated from farms. Overall, this assay is a promising test for viable pathogens detection and biosafety control.

Development of a new PCR assay and a recombinase-aided amplification based isothermal amplification coupled with lateral flow dipstick assay for potato late blight detection

Late blight caused by Phytophthora infestans is the most devastating potato disease, yet leaf infections are not reliably detected by current assays during the incubation phase of the pathogen and asymptomatic plants. In this study, the genome of P. infestans was queried against the NCBI nucleic acid database and a 1050 bp species-specific DNA fragment was identified. Using this fragment, polymerase chain reaction (PCR) and recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assays were developed for the detection of potato late blight pathogen. Both assays effectively distinguished P. infestans from related species (e.g., P. capsici). The detection limits of the three PCR primer pairs under optimized conditions (59.3 °C, 35 PCR cycles) were 1.31 × 10−1 fg, 1.18 × 10−2 fg, and 1.24 × 10−1 fg, corresponding to 27.31 copies, 2.49 copies, and 25.51 copies, respectively. While the detection limit of the RAA-LFD assay at 37 °C for 25 min was 5 × 10−4 fg (0.10 copies), which was better than all reported molecular detection assays for potato late blight. Both PCR (59.3 °C, 35 PCR cycles) and RAA-LFD (37 °C for 10 min) positively detected all symptomatic leaves. When extending the amplification time to 20 min, the RAA-LFD assay detected most of the leaves with incubation phase of the disease. The molecular detection assays developed in this study were species-specific and are expected to be used in detecting potato late blight in the incubation phase in leaves and asymptomatic plants, which will aid in accurate monitoring and early warning of potato late blight.

Clonally expanded HIV-1 proviruses with 5'-leader defects can give rise to nonsuppressible residual viremia.

BackgroundAntiretroviral therapy (ART) halts HIV-1 replication, decreasing viremia to below the detection limit of clinical assays. However, some individuals experience persistent nonsuppressible viremia (NSV) originating from CD4+ T cell clones carrying infectious proviruses. Defective proviruses represent over 90% of all proviruses persisting during ART and can express viral genes, but whether they can cause NSV and complicate ART management is unknown.MethodsWe undertook an in-depth characterization of proviruses causing NSV in 4 study participants with optimal adherence and no drug resistance. We investigated the impact of the observed defects on 5'-leader RNA properties, virus infectivity, and gene expression. Integration-site specific assays were used to track these proviruses over time and among cell subsets.ResultsClones carrying proviruses with 5'-leader defects can cause persistent NSV up to approximately 103 copies/mL. These proviruses had small, often identical deletions or point mutations involving the major splicing donor (MSD) site and showed partially reduced RNA dimerization and nucleocapsid binding. Nevertheless, they were inducible and produced noninfectious virions containing viral RNA, but lacking envelope.ConclusionThese findings show that proviruses with 5'-leader defects in CD4+ T cell clones can give rise to NSV, affecting clinical care. Sequencing of the 5'-leader can help in understanding failure to completely suppress viremia.FundingOffice of the NIH Director and National Institute of Dental and Craniofacial Research, NIH; Howard Hughes Medical Institute; Johns Hopkins University Center for AIDS Research; National Institute for Allergy and Infectious Diseases (NIAID), NIH, to the PAVE, BEAT-HIV, and DARE Martin Delaney collaboratories.

Hypothyroidism impairs development of the gastrointestinal tract in the ovine fetus.

Growth and maturation of the fetal gastrointestinal tract near term prepares the offspring for the onset of enteral nutrition at birth. Structural and functional changes are regulated by the prepartum rise in cortisol in the fetal circulation, although the role of the coincident rise in plasma tri-iodothyronine (T3) is unknown. This study examined the effect of hypothyroidism on the structural development of the gastrointestinal tract and the activity of brush-border digestive enzymes in the ovine fetus near term. In intact fetuses studied between 100 and 144days of gestation (dGA; term ∼145days), plasma concentrations of T3, cortisol and gastrin; the mucosal thickness in the abomasum, duodenum, jejunum and ileum; and intestinal villus height and crypt depth increased with gestational age. Removal of the fetal thyroid gland at 105-110 dGA suppressed plasma thyroxine (T4) and T3 concentrations to the limit of assay detection in fetuses studied at 130 and 144 dGA, and decreased plasma cortisol and gastrin near term, compared to age-matched intact fetuses. Hypothyroidism was associated with reductions in the relative weights of the stomach compartments and small intestines, the outer perimeter of the intestines, the thickness of the gastric and intestinal mucosa, villus height and width, and crypt depth. The thickness of the mucosal epithelial cell layer and muscularis propria in the small intestines were not affected by gestational age or treatment. Activities of the brush border enzymes varied with gestational age in a manner that depended on the enzyme and region of the small intestines studied. In the ileum, maltase and dipeptidyl peptidase IV (DPPIV) activities were lower, and aminopeptidase N (ApN) were higher, in the hypothyroid compared to intact fetuses near term. These findings highlight the importance of thyroid hormones in the structural and functional development of the gastrointestinal tract near term, and indicate how hypothyroidism in utero may impair the transition to enteral nutrition and increase the risk of gastrointestinal disorders in the neonate.

Serum High-Sensitivity Cardiac Troponin T as an Independent Predictor for Incident Coronary Heart Disease in the Japanese General Population: The Circulatory Risk in Communities Study (CIRCS).

Epidemiological evidence is extremely limited about high-sensitivity cardiac troponin T (hs-cTnT) and future coronary heart disease (CHD) events for the general population in countries with low mortality from CHD. Therefore, we investigated the association between hs-cTnT levels and the risk of incident CHD using a nested case-control study in a large Japanese cohort study. The participants were residents of four Japanese communities in the Circulatory Risk in Communities Study (CIRCS). We obtained 120 cases (81 men and 39 women, aged 38-86 years at baseline) of first incident CHD and 240 controls matched by age, sex, communities, and blood sampling term. Serum hs-cTnT levels were measured using an electrochemiluminescence immunoassay with stored sera collected between 2001 and 2011. The median period between sampling at survey and CHD incidence was 2.0 (interquartile range, 0.9-3.7) years. After adjusting for conventional risk factors, the multivariable odds ratios (ORs) of CHD were calculated using conditional logistic regression analyses. hs-cTnT ranged from ≤ 3 (assay detection limit) to 155 ng/L. Compared with the lowest quartile of hs-cTnT, multivariable ORs (95% confidence intervals) of CHD for the second, third, and highest quartiles were 1.30 (0.57-2.95), 2.48 (1.09-5.64), and 3.01 (1.27-7.12), respectively. Similar associations were observed after adjusting for estimated glomerular filtration, or after excluding matched groups, including people with chronic kidney disease. Serum hs-cTnT could predict CHD in the Japanese general population. These findings implicate a benefit from monitoring hs-cTnT to predict CHD even among populations in countries with low mortality from CHD.

A Healthy Vaginal Microbiota Remains Stable during Oral Probiotic Supplementation: A Randomised Controlled Trial.

The primary objective of this randomised, placebo-controlled, triple-blind study was to assess whether orally consumed Lactobacillus acidophilus La-14 (La-14) and Lacticaseibacillus rhamnosus HN001 (HN001) colonise a healthy human vagina. Furthermore, potential effects on vaginal microbiota and immune markers were explored. Fifty women devoid of vaginal complaints (Nugent score 0-3 and vaginal pH ≤ 4.5) were randomised into a 2-week intervention with either La-14 and HN001 as the verum product or a comparable placebo. Vaginal swab samples were collected at baseline, after one and two weeks of intervention, and after a one-week follow-up, for assessing colonisation of the supplemented lactobacilli, vaginal microbiota, and six specific immune markers. Colonisation of L. acidophilus and L. rhamnosus was not observed above the assay detection limit (5.29 and 5.11 log 10 genomes/swab for L. acidophilus and L. rhamnosus, respectively). Vaginal microbiotas remained stable and predominated by lactobacilli throughout the intervention, and vaginal pH remained optimal (at least 90% of participants in both groups had pH 4.0 or 4.5 throughout the study). Immune markers elafin and human β-defensin 3 (HBD-3) were significantly decreased in the verum group (p = 0.022 and p = 0.028, respectively) but did not correlate with any microbiota changes. Adverse events raised no safety concerns, and no undesired changes in the vaginal microbiota or immune markers were detected.

The Development and Characterization of a Highly Sensitive Mature TGFβ3 Assay to Evaluate Anti-TGFβ3 Target Engagement.

MTBT 1466A is a monoclonal antibody designed to bind to mature human TGFβ3 in human tissue and systemic circulation. To evaluate binding of this therapeutic, a mature TGFβ3 assay was needed to be able to monitor pharmacodynamic responses in non-human primate (NHP) studies. However, mature TGFβ3 levels in systemic circulation are very low and require development of a highly sensitive assay for detection. This study describes the development of a highly sensitive, drug-tolerant pharmacodynamic biomarker assay for demonstrating target engagement in a pre-clinical study using MTBT1466A. Since mature TGFβ3 is a dimer, a single MAb was used as both the capture and detection antibodies. This assay was developed on the SMCxPRO platform and qualified based on current accepted criteria for biomarker assays. The assay demonstrated specificity to mature TGFβ3, with a lower limit of quantification of 31.3pg/mL. Although baseline levels of mature TGFβ3 were below the assay detection limit in 40% of animals within our study, 2- to 16-fold increases were observed in many of the animals following multiple-dosing regimen.

Molecular diagnostics for monitoring insecticide resistance in the western flower thrips Frankliniella occidentalis.

Insecticide resistance has emerged in various western flower thrips (WFT) populations across the world, threatening the efficiency of chemical control applications. Elucidation of insecticide resistance mechanisms at the molecular level provides markers for the development of diagnostics to monitor the trait and support evidence-based resistance management. TaqMan and Droplet Digital polymerase chain reaction (ddPCR) diagnostics were developed and validated, against Sanger sequencing, in individual and pooled WFT samples respectively, for the G275E mutation (nicotinic acetylcholine receptor α6 gene, nAChR α6) associated with resistance to nAChR allosteric modulators, site I (spinosyns); L1014F, T929I, T929C and T292V mutations (voltage-gated sodium channel gene, vgsc) linked with pyrethroid resistance; and I1017M (chitin synthase 1 gene, chs1) conferring resistance to growth inhibitors affecting CHS1 (benzoylureas). The detection limits of ddPCR assays for mutant allelic frequencies (MAF) were in the range of 0.1%-0.2%. The assays were applied in nine WFT field populations from Crete, Greece. The G275E (MAF=29.66%-100.0%), T929I and T929V (combined MAF=100%), L1014F (MAF=11.01%-37.29%), and I1017M (MAF=17.74%-51.07%) mutations were present in all populations. The molecular diagnostics panel that was developed in this study can facilitate the quick and sensitive resistance monitoring of WFT populations at the molecular level, to support evidence-based insecticide resistance management strategies. © 2022 Society of Chemical Industry.

Rapid and simple colorimetric detection of quiescent Colletotrichum in harvested fruit using reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) technology

Anthracnose, caused by the fungus Colletotrichum gloeosporioides, is one of the major causes of postharvest decay of fruits and vegetables. Detection of the pathogen at an early stage of infection is crucial to developing a disease management strategy. In this work, a loop-mediated isothermal amplification (LAMP) assay was developed for the rapid detection of C. gloeosporioides targeting the transcript enoyl-CoA hydratase (ECH) that significantly upregulates only during C. gloeosporioides quiescent stage. The assay enabled a naked-eye detection of C. gloeosporioides RNA within 23 min based on a color change of LAMP products from pink to yellow. The detection limit of the LAMP assay was 1 pg of total RNA extracted from fruit peel in a 25 μL reaction. Positive results were obtained only in samples carrying the ECH gene, whereas no cross-reaction was observed for a different quiescent marker (histone deacetylase (HDAC)) or an appressorium marker (scytalone dehydratase, (SD)), indicating the high specificity of the method. Hence, the results indicate that the developed LAMP assay is a rapid, highly sensitive, and specific tool for the early detection of quiescent C. gloeosporioides and could be employed to manage postharvest diseases.

Specific biomarker mining and rapid detection of Burkholderia cepacia complex by recombinase polymerase amplification.

To mine specific proteins and their protein-coding genes as suitable molecular biomarkers for the Burkholderia cepacia Complex (BCC) bacteria detection based on mega analysis of microbial proteomic and genomic data comparisons and to develop a real-time recombinase polymerase amplification (rt-RPA) assay for rapid isothermal screening for pharmaceutical and personal care products. We constructed an automatic screening framework based on Python to compare the microbial proteomes of 78 BCC strains and 263 non-BCC strains to identify BCC-specific protein sequences. In addition, the specific protein-coding gene and its core DNA sequence were validated in silico with a self-built genome database containing 158 thousand bacteria. The appropriate methodology for BCC detection using rt-RPA was evaluated by 58 strains in pure culture and 33 batches of artificially contaminated pharmaceutical and personal care products. We identified the protein SecY and its protein-coding gene secY through the automatic comparison framework. The virtual evaluation of the conserved region of the secY gene showed more than 99.8% specificity from the genome database, and it can distinguish all known BCC species from other bacteria by phylogenetic analysis. Furthermore, the detection limit of the rt-RPA assay targeting the secY gene was 5.6 × 102 CFU of BCC bacteria in pure culture or 1.2 pg of BCC bacteria genomic DNA within 30 min. It was validated to detect <1 CFU/portion of BCC bacteria from artificially contaminated samples after a pre-enrichment process. The relative trueness and sensitivity of the rt-RPA assay were 100% in practice compared to the reference methods. The automatic comparison framework for molecular biomarker mining is straightforward, universal, applicable, and efficient. Based on recognizing the BCC-specific protein SecY and its gene, we successfully established the rt-RPA assay for rapid detection in pharmaceutical and personal care products.

In vitro anti-tumoral activity of two versatile cationic porphyrins on melanoma cells

Porphyrin (Por) dyes are considered photoactive entities with potential properties to be applied as photosensitizers (PS) in cancer Photodynamic Therapy (PDT). The use of suitable units, like pyridinium ones, is an important strategy to add peripheral and non-peripheral positive charges in the Por structure, and in that way develop effective cationic PSs for melanoma treatments. In this context, free-base porphyrins bearing thiopyridinium (1) or methoxypyridinium (2) units were studied on melanoma cells, and their PDT effectiveness was studied and compared. The different charge positions of the cationic peripheral units on the Por macrocycle contribute differently to their PDT behavior. The obtained results demonstrate high in vitro PDT efficacy for both PSs. For the highest PS concentration tested (20 [Formula: see text]M) the photocytotoxicity reaches the detection limit of the MTT assay upon 201 seconds of blue light irradiation ([Formula: see text] = 405 ± 20 nm) at an irradiance of 24.9 mW/cm2, which corresponds to a light dose of 5 J/cm2. Interestingly, under the same experimental conditions, cationic Por 1 shows very interesting PDT results at lower concentrations of 1 to 10 [Formula: see text]M.

Exceptional peroxidase-like activity of an iron and copper based organic framework nanosheet for consecutive colorimetric biosensing of glucose and kanamycin in real food samples.

Two-dimensional metal-organic framework nanosheets are attractive as peroxidase mimicking nanocatalysts due to their rich chemical functional groups, large surface area, high porosity, and accessible active sites. In this study, we synthesized FeCu bifunctional 2D MOF nanosheets using a solvothermal method. Fe and Cu ions were added as metal precursors, while organic amine and acid served as the organic ligands to construct the FeCu-MOF nanosheets. These nanosheets demonstrated robust peroxidase-like catalytic activities and were employed to develop a visual detection system for multiple targets, such as glucose and kanamycin. In the detection mechanism, glucose was oxidized into gluconic acid by glucose oxidase (GOx), leading to the generation of H2O2. When H2O2 is present, the FeCu-MOF NSs demonstrate high intrinsic peroxidase-like activity, which might catalytically oxidize 3,3',5,5'-tetramethylbenzidine (TMB) into a blue-coloured oxTMB product with a strong UV absorption at 654 nm. Subsequently, kanamycin was added to the above sensing system. The kanamycin strongly interacted with the FeCu-MOF NSs through H-bonding and blocked electron transfer, resulting in a colour change of the solution from blue to colourless with a weak UV absorption at 654 nm. Under the optimal conditions, the proposed colorimetric sensor exhibits an excellent linear response to glucose and kanamycin over the 0.25-5 μM and 0.02-0.1 μM ranges, respectively. The proposed colorimetric assay detection limits for glucose and kanamycin were found to be as low as 0.1 μM and 8 nM, respectively, and such a sensor shows excellent selectivity and sensitivity against different potential interferents. Thus, our proposed colorimetric assay was satisfactory when applied to glucose and kanamycin detection in agricultural and livestock husbandry samples.

Development of a TaqMan minor groove binding probe-based quantitative reverse transcription polymerase chain reaction for the detection and quantification of Zika virus.

Zika virus (ZIKV) infection has emerged as a global health concern following epidemic outbreaks of severe neurological disorders reported in Pacific and Americas since 2016. Therefore, a rapid, sensitive and specific diagnostic test for ZIKV infection is critical for the appropriate patient management and the control of disease spread. A TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed based on the conserved sequence regions of 463 ZIKV NS2B genes. The designed ZIKV qRT-PCR assay was evaluated for its detection limit, strain coverage and cross-reactivity. We further assessed the clinical applicability of qRT-PCR assay for ZIKV RNA detection using a total 18 simulated clinical specimens. The detection limit of the qRT-PCR assay was 11.276 ZIKV RNA copies at the 95% probability level (probit analysis, p<= 0.05). Both Asian and African ZIKV strains were detected by the qRT-PCR assay without cross-reacting with DENV-1, DENV-2, DENV-3, DENV-4, CHIKV, JEV, LGTV, GETV and SINV. The qRT-PCR assay demonstrated a perfect agreement (k = 1.000, P < 0.001) with the reference assay; the sensitivity and specificity of the qRT-PCR assay were 100% (95% CI= 79.6-100) and 100% (95% CI= 43.9-100) respectively. The qRT-PCR assay developed in this study is a useful diagnostic tool for the broad coverage detection and quantification of both the Asian and African ZIKV strains.

Development and evaluation of a rapid, specific, and sensitive loop-mediated isothermal amplification assay to detect Tenacibaculum sp. strain pbs-1 associated with black-spot shell disease in Akoya pearl oysters

Black-spot shell disease decreases pearl quality and threatens pearl oyster survival. Establishment of a rapid, specific, and sensitive assay to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease is of commercial importance. We developed a rapid, specific, and highly sensitive loop-mediated isothermal amplification (LAMP) assay to detect Tenacibaculum sp. Pbs-1 in Akoya pearl oysters Pinctada fucata. A set of five specific primers (two inner, two outer, and a loop) were designed based on the 16S-23S internal spacer region of strain Pbs-1. The optimum reaction temperature was 63°C, and concentrations of the inner and loop primers were 1.4 and 1.0µM, respectively. The LAMP product can be detected using agarose gel electrophoresis, and the color change in the reaction tube can be detected visually (by the naked eye) following the addition of malachite green. Our assay proved to be specific for strain Pbs-1, with no cross-reactivity with five other species of Tenacibaculum. The detection limit of the LAMP assay at 35min is 50pg, and at 60min it is 5fg. We evaluated the LAMP assay using diseased and healthy pearl oysters. The results demonstrate the suitability and simplicity of this test for rapid field diagnosis of strain Pbs-1.

A Simplified Multiplex PCR Assay for Simultaneous Detection of Six Viruses Infecting Diverse Chilli Species in India and Its Application in Field Diagnosis

Chilli is infected by at least 65 viruses globally, with a mixed infection of multiple viruses leading to severe losses being a common occurrence. A simple diagnostic procedure that can identify multiple viruses at once is required to track their spread, initiate management measures and manage them using virus-free planting supplies. The present study, for the first time, reports a simplified and robust multiplex PCR (mPCR) assay for the simultaneous detection of five RNA viruses, capsicum chlorosis orthotospovirus (CaCV), chilli veinal mottle virus (ChiVMV), large cardamom chirke virus (LCCV), cucumber mosaic virus (CMV), and pepper mild mottle virus (PMMoV), and a DNA virus, chilli leaf curl virus (ChiLCV) infecting chilli. The developed mPCR employed six pairs of primer from the conserved coat protein (CP) region of the respective viruses. Different parameters viz., primer concentration (150-450 nM) and annealing temperature (50 °C), were optimized in order to achieve specific and sensitive amplification of the target viruses in a single reaction tube. The detection limit of the mPCR assay was 5.00 pg/µL to simultaneously detect all the target viruses in a single reaction, indicating a sufficient sensitivity of the developed assay. The developed assay showed high specificity and showed no cross-amplification. The multiplex PCR assay was validated using field samples collected across Northeast India. Interestingly, out of 61 samples collected across the northeastern states, only 22 samples (36%) were positive for single virus infection while 33 samples (54%) were positive for three or more viruses tested in mPCR, showing the widespread occurrence of mixed infection under field conditions. To the best of our knowledge, this is the first report on the development and field validation of the mPCR assay for six chilli viruses and will have application in routine virus indexing and virus management.

Multiplex high-resolution melting assay for simultaneous detection of five key bacterial pathogens in urinary tract infections: A pilot study.

The diagnosis of urinary tract infections (UTIs) is usually based on the results of urine culture, but it is time-consuming, labor-intensive and has a low sensitivity. The aim of this study was to develop multiplex high-resolution melting assay (MHRM) for the simultaneous detection of five common bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Staphylococcus saprophyticus, Enterococcus faecalis, and group B streptococci (GBS)) directly from urine samples. A total of 287 urine specimens were evaluated by HRM assay and the results were compared with the conventional culture method. Five different melt curves generated and differentiated five bacterial pathogens. The detection limit of the MHRM assay was 1.5 × 103 CFU/ml for E. coli and K. pneumoniae and 1.5 × 102 CFU/ml for S. saprophyticus, E. faecalis and GBS. Compared to culture, the specificity of the MHRM assay ranged from 99.3 to 100%, and sensitivity 100% for all test pathogens. The MHRM assay developed in the current study might be functional tool for the diagnosis of UTIs and has the potential for direct detection of the organism in the clinical samples. Additionally, it creates results in less than 5 h, helping clinicians to start treatment with appropriate antimicrobial agents. This method could be a useful supplement to urine culture.

Development and application of multiple polymerase spiral reaction (PSR) assays for rapid detection of methicillin resistant Staphylococcus aureus and toxins from rice and flour products

Methicillin-Resistant Staphylococcus aureus (MRSA) is an important foodborne pathogen and produces a variety of toxins causing serious illnesses. This study aimed at developing a rapid detection assay for MRSA and toxins targeting on two housekeeping genes (mecA and femA) and 5 virulence factors (sea, seb, sec, see and pvl) using the polymerase spiral reaction (PSR), compared with PCR. For naked-eye result inspection, calcein was added and the PSR reaction can be completed at 65ᵒC within 60 min. The PSR assay was subjected to optimization, evaluation, and application in 3 different types of food sample made from rice and flour, designated rice and flour product. The limit of detection of PSR assay for the mecA, femA, sea, seb, sec, see and pvl genes was 4.2 pg/μL, 0.42 pg/μL, 630 pg/μL, 63 pg/μL, 12.3 pg/μL, 1.015 pg/μL and 53 pg/μL, respectively, which was higher than the sensitivity of PCR. In rice and flour products, the detection limit of PSR assay was 104 CFU/mL for mecA, and 103 CFU/mL for femA, sea, seb, sec, see and pvl genes. In conclusion, optimized PSR assay serves as an efficient tool for rapid, cost-effective and accurate testing of MRSA and is suitable for point-of-care detection.

Development of a visual multiplex fluorescent LAMP assay for the detection of foot-and-mouth disease, vesicular stomatitis and bluetongue viruses.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can be used to amplify target genes at a constant temperature, and it has several advantages, including convenience, specificity and sensitivity. However, due to the special interpretation methods of this technology for reaction results, all the previously reported LAMP detection methods have been restricted to identifying a single target, which limits the application of this technology. In this study, we modified conventional LAMP to include a quencher-fluorophore composite probe complementary to the F1c segment of the inner primer FIP; upon strand separation, a gain in the visible fluorescent signal was observed. The probes could be labeled with different fluorophores, showing different colors at the corresponding wavelengths. Therefore, this multiplex LAMP (mLAMP) assay can simultaneously detect 1-3 target sequences in a single LAMP reaction tube, and the results are more accurate and intuitive. In this study, we comprehensively demonstrated a single-reaction mLAMP assay for the robust detection of three cattle viruses without nonspecific amplification of other related pathogenic cattle viruses. The detection limit of this mLAMP assay was as low as 526-2477 copies/reaction for the recombinant plasmids. It is expected that this mLAMP assay can be widely used in clinical diagnosis.