Single qPCR Research Articles

The Challenge of Developing a Test to Differentiate Actinobacillus pleuropneumoniae Serotypes 9 and 11

Actinobacillus pleuropneumoniae is a major swine pathogen, classified into 19 serotypes based on capsular polysaccharide (CPS) loci. This study aimed to improve the diagnostic method to differentiate between serotypes 9 and 11, which are challenging to distinguish using conventional serological and molecular methods. A novel qPCR assay based on locked nucleic acid (LNA) probes was developed and validated using a collection of reference strains representing all known 19 serotypes. The assay demonstrated specificity in detecting the nucleotide variation characteristic of the serotype 9 reference strain. However, the analysis of a clinical isolate collection identified discrepancies between LNA-qPCR and serological results, prompting further investigation of the cps and O-Ag loci. Subsequent nanopore sequencing and whole-genome sequencing of a collection of 31 European clinical isolates, previously identified as serotype 9, 11, or undifferentiated 9/11, revealed significant genetic variations in the cps and O-Ag loci. Ten isolates had a cpsF sequence identical to that of the serotype 11 reference strain, while six isolates had single-nucleotide polymorphisms that were unlikely to cause significant coding changes. In contrast, 15 isolates had interruptions in the cpsF gene, distinct from that found in the serotype 9 reference strain, potentially leading to a serotype 9 CPS structure. In the O-Ag loci, differences between serotypes 9 and 11 were minimal, although some isolates had mutations potentially affecting O-Ag expression. Overall, these findings suggest that multiple genetic events can lead to the formation of a serotype 9 CPS structure, hindering the development of a single qPCR assay capable of detecting all cpsF gene mutations. Our results suggest that, currently, a comprehensive analysis of the cpsF gene is necessary to accurately determine whether the capsule of an isolate corresponds to serotype 9 or 11. Although such analyses are feasible with the advent of third-generation sequencing technologies, their accessibility, cost, and time to result limit their use in routine diagnostic applications. Under these circumstances, the designation of the hybrid serovar 9/11 remains a valid approach.

Advancing quantitative PCR with color cycle multiplex amplification.

Quantitative PCR (qPCR) is the gold standard for detection and quantitation of known DNA targets, but the scarcity of spectrally distinct fluorophores and filter sets limits the number of detectable targets. Here, we introduce color cycle multiplex amplification (CCMA) to significantly increase the number of detectable DNA targets in a single qPCR reaction using standard instrumentation. In CCMA, presence of one DNA target species results in a pre-programmed pattern of fluorescence increases. This pattern is distinguished by cycle thresholds (Cts) through rationally designed delays in amplification. For example, we design an assay wherein Staphylococcus aureus sequentially induces FAM, then Cy5.5, then ROX fluorescence increases with more than 3 cycles between each signal. CCMA offers notably higher potential for multiplexing because it uses fluorescence permutation rather than combination. With 4 distinct fluorescence colors, CCMA theoretically allows the detection of up to 136 distinct DNA target sequences using fluorescence permutation. Experimentally, we demonstrated a single-tube qPCR assay screening 21 sepsis-related bacterial DNA targets in samples of blood, sputum, pleural effusion and bronchoalveolar lavage fluid, with 89% clinical sensitivity and 100% clinical specificity, showing its potential as a powerful tool for advanced quantitative screening in molecular diagnostics.

Development of a Multiplex Real-Time Quantitative PCR Assay for Detecting Vaginal Microbiota in Chinese Women - China, 2021-2022.

The Nugent score, limited by subjectivity and personnel requirements, lacks accuracy. Establishing a precise and simple molecular test is therefore essential for detecting vaginal microbiota compositions and evaluating vaginal health. We evaluated the vaginal health of Chinese women using quantitative polymerase chain reaction (qPCR) to target Lactobacillus crispatus (L. crispatus), L. iners, Gardnerella vaginalis (G. vaginalis), Atopobium vaginae (A. vaginae), and Megasphaera phylotype1. bacterial vaginosis (BV)-related bacteria shared a fluorescent channel. Using 16S rDNA sequencing as a reference standard, we evaluated and validated the diagnostic accuracy of the qPCR assay. Both qPCR and 16S rDNA sequencing demonstrated 90.5% concordance in segregating vaginal community state type (CST), as visualized through heatmaps and PCoA. Spearman's correlation analysis revealed strong correlations between the two methods in calculating the RA of L. crispatus (CST I), L. iners (CST III), and BV-related bacteria (CST IV), with coefficients of 0.865, 0.837, and 0.827, respectively. Receiver operating characteristic analysis showed that qPCR had significant diagnostic accuracy for CST I, CST III, and CST IV (molecular BV), with area under the curve values of 0.967, 0.815, and 0.950, respectively, indicating strong predictive power. Vaginal health can be evaluated using a single qPCR amplification experiment, making the multiplex qPCR assay a highly accurate tool for this purpose.

Interlaboratory study on real-time PCR detection and quantification of the European anglerfish, pike, and seabream parvalbumin gene

This study presents a large-scale interlaboratory comparison (ILC) aimed at detecting and quantifying DNA from two European anglerfish (Lophius budegassa, Lophius piscatorius), pike (Esox lucius) and sea bream (Spondyliosoma cantharus) using real-time qPCR. To detect amplification of the parvalbumin genetic marker, single and multiplex qPCR assays using EvaGreen® dye or TaqMan™ probes were used. Genomic DNA isolated from target fish species and an advanced DNA calibrator, gBlocks® gene fragments, were used as standards. The DNA of anglerfish, pike and sea bream as well as their mixtures were analysed together with 14 other non-target fish species. All target fish samples were correctly identified by the participating laboratories. Qualitative assessment of anglerfish and seabream DNA showed an accuracy rate of 100%, while pike DNA achieved a match rate of 99%. Validation of quantitative protocols in four different laboratories consistently achieved z-scores below 2, indicating satisfactory performance and confirming the high degree of similarity of laboratory results. Furthermore, high accuracy and efficiency were demonstrated for the quantification of anglerfish and seabream DNA by triplex qPCR using TaqMan™ probes. Regarding the selected gene marker, the major fish allergenic protein parvalbumin enables indirect detection and quantification of the allergen in the sample. Therefore, the use of proposed protocols can significantly contribute to protecting the health of consumers and to controlling the food market.Graphical abstract

Abstract 2428: Novel ctDNA technology for early cancer detection by immunoprecipitation of tumor associated ctDNA fragments and analysis by qPCR

Abstract Introduction: Current ctDNA methods involve extraction of all plasma cfDNA, next generation sequencing (NGS) and complex bioinformatic analysis. We have developed a novel ctDNA analysis in which classes of tumor derived ctDNA are isolated, with removal of all non-tumor cfDNA of the same sequences, for analysis by qPCR. We now report the results of this analysis on cohorts of patients diagnosed with common solid cancers. Methods: We developed a chromatin immunoprecipitation (ChIP)/qPCR method for the isolation of cell free plasma CTCF-DNA (cfCTCF-DNA) nucleoproteins that include binding sites selectively occupied in cancer. CfDNA fragments of non-cancer origin that comprise the same CTCF binding site sequences are nucleosome covered and washed away. We performed plasma CTCF ChIP-Seq discovery experiments to identify CTCF gain of occupancy cancer biomarker sequences by analysis of plasma samples taken from cancer patients as well as patients with inflammatory conditions and healthy volunteers. Suitable pan-cancer, or cancer tissue specific, biomarkers were selected as characterized CTCF binding site sequences that were present in the ChIP isolates of cancer patients but absent in isolates from patients without cancer. We then developed qPCR assays for selected CTCF gain of occupancy biomarker sequences. We initially investigated whether CTCF gain of occupancy biomarkers would identify patients with cancer in a small cohort of subjects with acute myeloid leukemia (AML) (n=31), breast (n=10), prostate (n=10), liver (n=10) or colorectal cancer (CRC) (n=13) and 50 control subjects including 15 subjects with an inflammatory condition. Results: In our small initial experiments, a simple cutoff where a qPCR result exceeding the cutoff was positive, a single qPCR assay detected 19 of 31 AML cases (61%) with 1 false positive result among 50 control samples (98% specificity). Addition of a second qPCR assay to make a 2-member qPCR panel, where a result exceeding cutoff in either or both assays was classified as positive, resulted in the detection of 23 of 31 AML cases (74%) with 2 false positive results (96% specificity). On the same basis a 2-member qPCR panel also detected 58% of solid cancers including 44% of stage I cases. Another 2-member qPCR panel detected 77% of CRC cases at 92% specificity. We now report the results of investigations of larger cohorts of patients with common solid cancers by cancer type, stage and tissue specificity. Conclusion: Combining the isolation of plasma cfCTCF-DNA nucleoproteins with PCR analysis of cancer associated CTCF gain of occupancy binding site sequences is a novel ctDNA analysis technology that may provide the basis of useful, rapid, low cost, automated liquid biopsy methods. Further clinical studies to ascertain the clinical accuracy of the method are required. Citation Format: Jacob Micallef, Dorian Pamart, Jean-Valery V. Turatsinze, Thomas Bygott, Benjamin Berman, Brieuc Cuvelier, Marielle Herzog. Novel ctDNA technology for early cancer detection by immunoprecipitation of tumor associated ctDNA fragments and analysis by qPCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2428.

Determining Pneumocystis jirovecii Colonisation from Infection Using PCR-Based Diagnostics in HIV-Negative Individuals.

Pneumocystis jirovecii pneumonia is increasingly diagnosed with highly sensitive PCR diagnostics in immunocompromised, HIV-negative individuals. We assessed the performance of our in-house quantitative PCR with the aim to optimise interpretation. Retrospective audit of all positive P. jirovecii qPCRs on induced sputum or BAL fluid at a single centre from 2012 to 2023. Medical and laboratory records were analysed and people with HIV were excluded. Cases were categorised as colonisation, high-probability PCP or uncertain PCP infection against a clinical gold standard incorporating clinico-radiological data. Quantitative PCR assay targeting the 5s gene was utilised throughout the time period. Of the 82 positive qPCRs, 28 were categorised as high-probability PCP infection, 30 as uncertain PCP and 24 as colonisation. There was a significant difference in qPCR values stratified by clinical category but not respiratory sample type. Current assay performance with a cutoff of 2.5 × 105 copies/mL had a sensitivity of 50% (95% CI, 30.65-69.35%) and specificity of 83.33% (95% CI, 62.62-95.26%). Youden Index calculated at 6.5 × 104 copies/mL had a sensitivity of 75% (56.64-87.32%, 95% CI) and specificity of 66.67% (46.71-82.03%, 95% CI). High and low cutoffs were explored. Significant variables associated with infection were age > 70 years old, the presence of fever, hypoxia or ground glass changes. A single qPCR cutoff cannot reliably determine P. jirovecii infection from colonisation. Low and high cutoffs are useful, however, a large "possible infection" cohort will remain where interpretation of clinic-radiological factors remains essential. Standardisation of assays with prospective validation in specific immunocompromised groups will allow greater generalisability and allow large-scale prospective assay validation to be performed.

A multiplex real-time RT-PCR system to simultaneously diagnose 16 pathogens associated with swine respiratory disease.

Swine respiratory disease (SRD) is a major disease complex in pigs that causes severe economic losses. SRD is associated with several intrinsic and extrinsic factors such as host health status, viruses, bacteria, and environmental factors. Particularly, it is known that many pathogens are associated with SRD to date, but most of the test to detect those pathogens can be normally investigated only one pathogen while taking time and labor. Therefore, it is desirable to develop rapidly and efficiently detectable methods those pathogens to minimize the damage caused by SRD. We designed a multiplex real-time RT-PCR (RT-qPCR) system to diagnose simultaneously 16 pathogens, including nine viruses and seven bacteria associated with SRD, on the basis of single qPCR and RT-qPCR assays reported in previous studies. Multiplex RT-qPCR system we designed had the same ability to single RT-qPCR without significant differences in detection sensitivity for all target pathogens at minimum to maximum genomic levels. Moreover, the primers and probes used in this system had highly specificity because the sets had not been detected pathogens other than the target and its taxonomically related pathogens. Furthermore, our data demonstrated that this system would be useful to detect a causative pathogen in the diagnosis using oral fluid from healthy pigs and lung tissue from pigs with respiratory disorders collected in the field. The rapid detection of infected animals from the herd using our system will contribute to infection control and prompt treatment in the field.

Environmental sampling for typhoidal Salmonellas in household and surface waters in Nepal identifies potential transmission pathways.

Salmonella Typhi and Salmonella Paratyphi, fecal-oral transmitted bacterium, have temporally and geographically heterogeneous pathways of transmission. Previous work in Kathmandu, Nepal implicated stone waterspouts as a dominant transmission pathway after 77% of samples tested positive for Salmonella Typhi and 70% for Salmonella Paratyphi. Due to a falling water table, these spouts no longer provide drinking water, but typhoid fever persists, and the question of the disease's dominant pathway of transmission remains unanswered. We used environmental surveillance to detect Salmonella Typhi and Salmonella Paratyphi A DNA from potential sources of transmission. We collected 370, 1L drinking water samples from a population-based random sample of households in the Kathmandu and Kavre Districts of Nepal between February and October 2019. Between November 2019 and July 2021, we collected 380, 50mL river water samples from 19 sentinel sites on a monthly interval along the rivers leading through the Kathmandu and Kavre Districts. We processed drinking water samples using a single qPCR and processed river water samples using differential centrifugation and qPCR at 0 and after 16 hours of liquid culture enrichment. A 3-cycle threshold (Ct) decrease of Salmonella Typhi or Salmonella Paratyphi, pre- and post-enrichment, was used as evidence of growth. We also performed structured observations of human-environment interactions to understand pathways of potential exposure. Among 370 drinking water samples, Salmonella Typhi was detected in 7 samples (1.8%) and Salmonella Paratyphi A was detected in 4 (1.0%) samples. Among 380 river water samples, Salmonella Typhi was detected in 171 (45%) and Salmonella Paratyphi A was detected in 152 (42%) samples. Samples located upstream of the Kathmandu city center were positive for Salmonella Typhi 12% of the time while samples from locations in and downstream were positive 58% and 67% of the time respectively. Individuals were observed bathing, washing clothes, and washing vegetables in the rivers. These results suggest that drinking water was not the dominant pathway of transmission of Salmonella Typhi and Salmonella Paratyphi A in the Kathmandu Valley in 2019. The high degree of river water contamination and its use for washing vegetables raises the possibility that river systems represent an important source of typhoid exposure in Kathmandu.

Deep Domain Adaptation Enhances Amplification Curve Analysis for Single-Channel Multiplexing in Real-Time PCR.

Data-driven approaches for molecular diagnostics are emerging as an alternative to perform an accurate and inexpensive multi-pathogen detection. A novel technique called Amplification Curve Analysis (ACA) has been recently developed by coupling machine learning and real-time Polymerase Chain Reaction (qPCR) to enable the simultaneous detection of multiple targets in a single reaction well. However, target classification purely relying on the amplification curve shapes currently faces several challenges, such as distribution discrepancies between different data sources of synthetic DNA and clinical samples (i.e., training vs testing). Optimisation of computational models is required to achieve higher performance of ACA classification in multiplex qPCR through the reduction of those discrepancies. Here, we proposed a novel transformer-based conditional domain adversarial network (T-CDAN) to eliminate data distribution differences between the source domain (synthetic DNA data) and the target domain (clinical isolate data). The labelled training data from the source domain and unlabelled testing data from the target domain are fed into the T-CDAN, which learns both domains' information simultaneously. After mapping the inputs into a domain-irrelevant space, T-CDAN removes the feature distribution differences and provides a clearer decision boundary for the classifier, resulting in a more accurate pathogen identification. Evaluation of 198 clinical isolates containing three types of carbapenem-resistant genes (blaNDM, blaIMP and blaOXA-48) illustrates a curve-level accuracy of 93.1% and a sample-level accuracy of 97.0% using T-CDAN, showing an accuracy improvement of 20.9% and 4.9% respectively, compared with previous methods. This research emphasises the importance of deep domain adaptation to enable high-level multiplexing in a single qPCR reaction, providing a solid approach to extend qPCR instruments' capabilities without hardware modification in real-world clinical applications.

Detection of Hazelnut and Almond Adulteration in Olive Oil: An Approach by qPCR.

Virgin olive oil (VOO), characterized by its unique aroma, flavor, and health benefits, is subject to adulteration with the addition of oils obtained from other edible species. The consumption of adulterated olive oil with nut species, such as hazelnut or almond, leads to health and safety issues for consumers, due to their high allergenic potential. To detect almond and hazelnut in olive oil, several amplification systems have been analyzed by qPCR assay with a SYBR Green post-PCR melting curve analysis. The systems selected were Cora1F2/R2 and Madl, targeting the genes coding the allergenic protein Cor a 1 (hazelnut) and Pru av 1 (almond), respectively. These primers revealed adequate specificity for each of the targeted species. In addition, the result obtained demonstrated that this methodology can be used to detect olive oil adulteration with up to 5% of hazelnut or almond oil by a single qPCR assay, and with a level as low as 2.5% by a nested-qPCR assay. Thus, the present research has shown that the SYBR-based qPCR assay can be a rapid, precise, and accurate method to detect adulteration in olive oil.

An improved method for determining frequency of multiple variants of SARS-CoV-2 in wastewater using qPCR assays

Wastewater-based surveillance has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription polymerase chain reaction (RT-PCR) or whole genome sequencing (WGS). Rapid, reliable RT-PCR assays continue to be needed to determine the relative frequencies of VOCs and sub-lineages in wastewater-based surveillance programs. The presence of multiple mutations in a single region of the N-gene allowed for the design of a single amplicon, multiple probe assay, that can distinguish among several VOCs in wastewater RNA extracts. This approach which multiplexes probes designed to target mutations associated with specific VOC's along with an intra-amplicon universal probe (non-mutated region) was validated in singleplex and multiplex. The prevalence of each mutation (i.e. VOC) is estimated by comparing the abundance of the targeted mutation with a non-mutated and highly conserved region within the same amplicon. This is advantageous for the accurate and rapid estimation of variant frequencies in wastewater. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from several communities in Ontario, Canada in near real time from November 28, 2021 to January 4, 2022. This includes the period of the rapid replacement of the Delta variant with the introduction of the Omicron variant in these Ontario communities in early December 2021. The frequency estimates using this assay were highly reflective of clinical WGS estimates for the same communities. This style of qPCR assay, which simultaneously measures signal from a non-mutated comparator probe and multiple mutation-specific probes contained within a single qPCR amplicon, can be applied to future assay development for rapid and accurate estimations of variant frequencies.

Abstract 788: Novel multiplex methylation-specific qPCR strategy for early CRC detection

Abstract Colorectal cancer (CRC) can be cured if detected early. Analysis of abnormalities in cfDNA methylation is an emerging and powerful strategy for early cancer detection. Recently, targeted methylation NGS methods have shown encouraging performance in CRC detection, but their high cost and complicated workflow can be challenging for many clinical settings. At the same time, the alternative, single methylation biomarker PCR assays, demonstrate limited sensitivity and specificity. An accurate, accessible and affordable assay ideal for clinical applications requires an easily scalable set-up and a set of high quality biomarkers providing sufficient predictive power. We aim to develop an assay fulfilling the CMS definitive performance guideline of LBx CRC screening – at least 74% sensitivity and 90% specificity. Here we report the development of a novel multiplex methylation-specific qPCR (MMS-qPCR) strategy for early CRC detection. Previously, we established detailed methylation profiles of our proprietary CRC targeted methylation sequencing panel with 560 biomarkers, based on more than 500 plasma samples of both healthy controls and CRC patients of various stages. We selected 9 biomarkers that are both highly sensitive and specific for CRC. These 9 markers show methylation signatures prevalent in patients and have very low noise in healthy controls. Along with one reference gene, the 9 CRC hypermethylated biomarkers can be multiplexed and analyzed in a single methylation-specific qPCR reaction. Analytical sensitivity and specificity were determined using a model system where hypermethylated DNA molecules were spiked into normal cell free DNA background at various percentages. With 10 ng of input, we can detect a single copy of the hypermethylated genome, achieving 0.006% limit of detection. Plasma samples from 10 stage I and 11 stage II CRC patients as well as 104 age-matched control subjects were subjected to the PCR analysis to yield a specificity of 96% and detection rate of 50% for stage I and 81% for stage II. The total turnaround time for analysis was <4 hours including DNA isolation, bisulfite conversion and qPCR analysis. The MMS-qPCR described here provides an accurate, accessible, and affordable liquid biopsy assay for the early detection of CRC. The same development strategy can also be applied to many other cancer types. Citation Format: Yun Bao, Heng Wang, Peter Vu, Grace Zhao, Shengrong Lin. Novel multiplex methylation-specific qPCR strategy for early CRC detection [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 788.

Optimization of a high-throughput nanofluidic real-time PCR to detect and quantify of 15 bacterial species and 92 Streptococcus pneumoniae serotypes

Sensitive tools for detecting concurrent colonizing pneumococcal serotypes are needed for detailed evaluation of the direct and indirect impact of routine pneumococcal conjugate vaccine (PCV) immunization. A high-throughput quantitative nanofluidic real-time PCR (Standard BioTools ‘Fluidigm’) reaction-set was developed to detect and quantify 92 pneumococcal serotypes in archived clinical samples. Nasopharyngeal swabs collected in 2009–2011 from South African children ≤ 5 years-old, previously serotyped with standard culture-based methods were used for comparison. The reaction-set within the ‘Fluidigm’ effectively amplified all targets with high efficiency (90–110%), reproducibility (R2 ≥ 0.98), and at low limit-of-detection (< 102 CFU/ml). A blind analysis of 1 973 nasopharyngeal swab samples showed diagnostic sensitivity > 80% and specificity > 95% compared with the referent standard, culture based Quellung method. The qPCR method was able to serotype pneumococcal types with good discrimination compared with Quellung (ROC-AUC: > 0.73). The high-throughput nanofluidic real-time PCR method simultaneously detects 57 individual serotypes, and 35 serotypes within 16 serogroups in 96 samples (including controls), within a single qPCR run. This method can be used to evaluate the impact of current PCV formulations on vaccine-serotype and non-vaccine-serotype colonization, including detection of multiple concurrently colonizing serotypes. Our qPCR method can allow for monitoring of serotype-specific bacterial load, as well as emergence or ongoing transmission of minor or co-colonizing serotypes that may have invasive disease potential.

Content Size-Dependent Alginate Microcapsule Formation Using Centrifugation to Eliminate Empty Microcapsules for On-Chip Imaging Cell Sorter Application.

Alginate microcapsules are one of the attractive non-invasive platforms for handling individual cells and clusters, maintaining their isolation for further applications such as imaging cell sorter and single capsule qPCR. However, the conventional cell encapsulation techniques provide huge numbers of unnecessary empty homogeneous alginate microcapsules, which spend an excessive majority of the machine time on observations and analysis. Here, we developed a simple alginate cell encapsulation method to form content size-dependent alginate microcapsules to eliminate empty microcapsules using microcapillary centrifugation and filtration. Using this method, the formed calcium alginate microcapsules containing the HeLa cells were larger than 20m, and the other empty microcapsules were less than 3m under 4000 rpm centrifugation condition. We collected cell-containing alginate microcapsules by eliminating empty microcapsules from the microcapsule mixture with simple one-step filtration of a 20 m cell strainer. The electrical surface charge density and optical permeability of those cell-encapsulated alginate microcapsules were also evaluated. We found that the surface charge density of cell-encapsulated alginate microbeads is more than double that of cells, indicating that less voltage is required for electrical cell handling with thin alginate gel encapsulation of samples. The permeability of the alginate microcapsule was not improved by changing the reflective index of the medium buffer, such as adding alginate ester. However, the minimized thickness of the alginate gel envelope surrounding cells in the microcapsules did not degrade the detailed shapes of encapsulated cells. Those results confirmed the advantage of alginate encapsulation of cells with the centrifugation method as one of the desirable tools for imaging cell sorting applications.

119 Single closed-tube qPCR assay with dual-labelled probes for improved sexing of equine embryos

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Dual phage amplification-mediated multiplex detection strategies for the simultaneous detection of Salmonella enterica and Staphylococcus aureus

Multiplex bacteria detection is essential to monitor various pathogens situations where multiple pathogen species are a concern. Herein, we explored novel dual phage amplification-based strategies (PAA) combined with a multiplex detection technique for simultaneous bacteria detection in one single tube. This assay was normalized on two common foodborne pathogens: Salmonella enterica and Staphylococcus aureus . In the presence of the target bacteria, phages could specifically recognize the viable cells and replicate themselves within the cells to produce more progeny phages. After thermal lysis within 10 min, the products with DNA of the progeny phages were analyzed with the multiplex detection strategies. The specificity of the multiplex detection strategies integrated with the high sensitivity of the PAA assay allowed this assay to be rapid, specific, and sensitive for simultaneous quantification of Salmonella and Staphylococcus aureus . Optimized parameters of PAA, single and multiplex qPCR were defined, and the analytical sensitivities, specificities, and stabilities of single and multiplex reactions were validated under defined experimental conditions and in real samples. The results demonstrated wide linear ranges (10–10 8 CFU/mL), with detection limits of 10 CFU/mL for all bacteria tested. Moreover, the total detection time could be reduced to no more than 4 h. This assay was successfully applied to real food samples consistent with the results comparable to traditional plate counting methods. Due to the rapid and simple operation, low sensitivity and cost, high specificity, and the ability to distinguish live bacteria, the proposed PAA-based multiplex qPCR assay provides an effective and promising strategy for the simultaneous detection of viable bacteria. • PAA relied on novel phages allows signal amplification to increase sensitivity. • PAA combined with single detection assay can achieve sensitive quantification for either Salmonella or Staphylococcus. • Dual PAA-mediated multiplex detection assay was applied for simultaneous detection. • This assay presented wide linear ranges of 10–10 8 CFU/mL, with the LODs of 10 CFU/mL for all bacteria tested.

Development of a TaqMan-based multiplex real-time PCR for simultaneous detection of four feline diarrhea-associated viruses.

Since their recent discovery, the prevalence of novel feline enteric viruses, including feline bocavirus 1 (FBoV-1), feline astrovirus (FeAstV), and feline kobuvirus (FeKoV), has been reported in China. Co-infections of these viruses with feline parvovirus (FPV) are common causes of diarrhea in cats. Viral co-infections are difficult to identify because of their non-specific clinical signs. To detect and identify these viruses, a quick and specific pathogen-testing approach is required. Here, we establish a real-time PCR (qPCR) based on multiple TaqMan probes for the simultaneous detection of FBoV-1, FeAstV, FeKoV, and FPV. Specific primers and TaqMan fluorescent probes were designed to ensure specificity. The results showed that the detection limit of single qPCR was up to 10 copies, and the detection limit of multiplex qPCR was up to 100 copies, with correlation coefficients >0.995 in all cases. Clinical sample detection revealed a 25.19% (34/135) total rate of co-infection among the viruses and a 1.48% (2/135) quadruple infection rate. Thus, this multiplex qPCR approach can serve as a quick, sensitive, and specific diagnostic tool for FBoV-1, FeAstV, FeKoV, and FPV identification, and it may be utilized for routine surveillance of these emerging and reemerging feline enteric viruses.

Development and validation of molecular methodologies for identification of biological traces from crime scenes

<h2>Abstract</h2> This study aimed to develop a methodology to identify biological fluids in sexual assault cases through mRNA markers. Biological fluid samples such as blood, saliva, and semen were collected from volunteers and submitted to RT-qPCR reactions with specific primers for the biomarkers HTN3 (saliva), ALAS (blood) andTGM4 (semen). The Melting (Tm) of each biological fluid was analyzed and the result inferred a high specificity capable of differentiating such traces. Biplex systems were generated to improve trace analysis in a single qPCR reaction.

Neuronally expressed PDL1, not PD1, suppresses acute nociception

PDL1 is a protein that induces immunosuppression by binding to PD1 expressed on immune cells. In line with historical studies, we found that membrane-bound PD1 expression was largely restricted to immune cells; PD1 was not detectable at either the mRNA or protein level in peripheral neurons using single neuron qPCR, immunolabeling and flow cytometry. However, we observed widespread expression of PDL1 in both sensory and sympathetic neurons that could have important implications for patients receiving immunotherapies targeting this pathway that include unexpected autonomic and sensory related effects. While signaling pathways downstream of PD1 are well established, little to no information is available regarding the intracellular signaling downstream of membrane-bound PDL1 (also known as reverse signaling). Here, we administered soluble PD1 to engage neuronally expressed PDL1 and found that PD1 significantly reduced nocifensive behaviors evoked by algogenic capsaicin. We used calcium imaging to examine the underlying neural mechanism of this reduction and found that exogenous PD1 diminished TRPV1-dependent calcium transients in dissociated sensory neurons. Furthermore, we observed a reduction in membrane expression of TRPV1 following administration of PD1. Exogenous PD1 had no effect on pain-related behaviors in sensory neuron specific PDL1 knockout mice. These data indicate that neuronal PDL1 activation is sufficient to modulate sensitivity to noxious stimuli and as such, may be an important homeostatic mechanism for regulating acute nociception.

Performance of NIST SRM® 2917 with 13 recreational water quality monitoring qPCR assays

Fecal pollution remains a significant challenge for recreational water quality management worldwide. In response, there is a growing interest in the use of real-time quantitative PCR (qPCR) methods to achieve same-day notification of recreational water quality and associated public health risk as well as to characterize fecal pollution sources for targeted mitigation. However, successful widespread implementation of these technologies requires the development of and access to a high-quality standard control material. Here, we report a single laboratory qPCR performance assessment of the National Institute of Standards and Technology Standard Reference Material 2917 (NIST SRM® 2917), a linearized plasmid DNA construct that functions with 13 recreational water quality qPCR assays. Performance experiments indicate the generation of standard curves with amplification efficiencies ranging from 0.95 ± 0.006 to 0.99 ± 0.008 and coefficient of determination values (R2) ≥ 0.980. Regardless of qPCR assay, variability in repeated measurements at each dilution level were very low (quantification threshold standard deviations ≤ 0.657) and exhibited a heteroscedastic trend characteristic of qPCR standard curves. The influence of a yeast carrier tRNA added to the standard control material buffer was also investigated. Findings demonstrated that NIST SRM® 2917 functions with all qPCR methods and suggests that the future use of this control material by scientists and water quality managers should help reduce variability in concentration estimates and make results more consistent between laboratories.