SYBR Green-based qPCR Research Articles

COMPARISON OF YIELD AND QUALITY OF CIRCULATING CELL-FREE DNA FROM K2EDTA AND K3EDTA COLLECTIONS IN HEALTHY SUBJECTS

Background: Circulating cell-free DNA (cfDNA) is a biomarker for various clinical applications, including detecting and monitoring cancer. However, blood collection tubes can affect the yield and quality of cfDNA. Since specific cfDNA collection tubes are costly, K2EDTA and K3EDTA anticoagulant tubes are alternatives in routine clinical laboratories. Objectives: This study aimed to compare the efficiency of cfDNA extraction from plasma collected in K2EDTA and K3EDTA tubes and evaluate implementation for molecular diagnostics. Methods: Blood samples from 38 healthy subjects were collected in K2EDTA and K3EDTA tubes that were processed within 2 hours. The extracted cfDNA was measured and performed using SYBR Green-based qPCR for three endogenous reference genes (GAPDH, HPRT1, TFRC). The cfDNA yield and the amplification efficiency of these genes were compared between K2EDTA and K3EDTA tubes using the Mann-Whitney U test. Results: There were no significant differences in cfDNA concentration between K2EDTA and K3EDTA tubes (p=0.051). However, qPCR analysis revealed significantly higher copy numbers of TFRC and HPRT1 in K2EDTA tubes than in K3EDTA tubes (p<0.05). No significant difference was found for GAPDH. Conclusion: The results indicate that K2EDTA and K3EDTA tubes are an alternative option for cfDNA analysis if samples are processed quickly after a blood draw, which offers flexibility and cost savings in resource-limited areas.

7397 Characterization of Sperm-Carried IGF2 Transcript Variants

Abstract Disclosure: R. Cannarella: None. M. Suryavanshi: None. A. Miller: None. S.D. Lundy: None. A.E. Calogero: None. Background: We have previously identified the expression of the highly-conserved paternally-imprinted insulin-like growth factor 2 (IGF2) gene in human spermatozoa and suggested its role in early embryo development. Although there are 5 different variants of the IGF2, there is no evidence of the variants expressed in human spermatozoa. Objective: The present basic research study was undertaken to characterize the IGF2 transcript variant(s) expressed in human spermatozoa and to develop a sensitive method that can distinguish variants based on quantitative polymerase chain reaction (qPCR). Methods: Two post-gradient sperm samples from healthy, fertile men with proven paternity were used. Granulosa cells (GCs) obtained after the oocyte retrieval procedure and subsequently isolated and purified from follicular fluid were used as controls. Each sample underwent RNA extraction, cDNA synthesis, and PCR. The PCR product was ligated with the pCR4-TOPO vector and used to transform TOPO10 one-shot electro-competent cells of E. coli DH-10α. For each sample, 32 white colonies (true positive clones selected by blue-white screening method) were grown per sample (total in 96). All 96 clones were fully characterized by Sanger sequencing and single nucleotide polymorphism (SNP) were detected. Variations in products from single IGF2 gene primers pair were recorded on Sybr green-based qPCR, such as variations in melting temperatures (Tm), product size, and number of copies. Results: We successfully isolated and identified 96 true positive colonies, which were confirmed as IGF2 cDNA clones. This comprehensive analysis revealed 11 distinct genetic variants within the cDNA clones. Specifically, these variants comprised 6 SNPs and 5 deletions, highlighting the diversity in the sequences. These findings were particularly significant in relation to the IGF2 primer employed in the study, as they indicated the presence of 11 different sequence variants. By Sybr green qPCR, clones derived from GCs and sperm samples predominantly showed an IGF2 transcript variant with Tms of 89.01°C, 88.86°C, and 88.71°C. In a surprising observation, the sperm samples exhibited three sperm-specific variants with a range of a Tm ranging from 89.01°C to 88.26°C. Furthermore, the deletions were characterized by a significantly lower Tm, ranging between 78.86°C and 71.11°C. Conclusion: The IGF2 transcript is intrinsically present in sperm samples, albeit with notable variations. The detected variants likely arise from SNPs within the transcripts and the deletions could be attributed to splicing activity during maturation or post-transcriptional modifications. To our knowledge, this is the first study targeting the IGF2 transcript variants in spermatozoa from fertile men. Basic research is needed to understand the implications of different sperm-specific IGF2 transcript variants on early embryo development. Presentation: 6/2/2024

Retrospective Detection of Ophidiomyces ophidiicola from Snake Moults Collected in Bieszczady Mountains, Poland.

Ophidiomyces ophidiicola, the causative agent of ophidiomycosis, poses a potential threat to wild snakes worldwide. This study aimed to retrospectively investigate the prevalence of O. ophidiicola in archived snake moults collected from the San River Valley in the Bieszczady Mountains, Poland, from 2010 to 2012. Using qPCR for O. ophidiicola detection and conventional PCR for clade characterisation, we analysed 58 moults and one road-killed specimen of Zamenis longissimus and Natrix natrix. A novel combination of primers (ITS2L) was used to simultaneously confirm SYBR Green-based qPCR results and perform genotyping. O. ophidiicola has been detected from two Z. longissimus and one N. natrix specimens. The identified clade (I-B) is consistent with those found in wild snakes of eastern Europe and San River Valley, indicating that O. ophidiicola has been present in this region for at least a decade. This study underscores the value of historical samples in understanding the long-term presence of pathogens and highlights the potential role of environmental reservoirs in the persistence of O. ophidiicola. Our findings are crucial for informing conservation strategies for the endangered Aesculapian snake populations in Poland, emphasising the need for ongoing monitoring and habitat management to mitigate the potential impact of ophidiomycosis.

Development of a duplex qPCR assay for the detection of coinfection in eels by Japanese eel endothelial cell-infecting virus and Anguillid herpesvirus-1

Japanese eel endothelial cell-infecting virus (JEECV) and Anguillid herpesvirus 1 (AngHV-1) are virulent pathogens in eel aquaculture farms in the Republic of Korea and coexist in the same host. To date, no diagnostic method has been developed that detects them simultaneously. We developed a duplex real-time polymerase chain reaction (duplex qPCR) assay to detect both JEECV and AngHV-1 simultaneously in a single reaction. Two sets of specific primers and TaqMan probes were designed to target the large T-antigen gene of JEECV and the DNA polymerase catalytic subunit gene of AngHV-1. The assay was optimized by changing the primer and probe concentrations and the annealing temperature of the reaction. The duplex qPCR could specifically amplify the specific genes of the two viruses with high efficiency and coefficient of determination. The assay could detect both viruses at a limit of detection of 8 fg/μL. The diagnostic performance of the developed assay was determined by analyzing 82 clinical samples (72 infected and 10 healthy) and comparing the results with those of conventional PCR and SYBR Green-based qPCR; the results correlated well with each other. The duplex qPCR showed 100% and 98.6% diagnostic sensitivity for JEECV and AngHV-1, respectively. The diagnostic specificity of this assay was validated using two potential viral strains, red sea bream iridovirus and Ostreid herpesvirus-1 microvariant, reported in the aquaculture industry. In contrast, the methods already developed including conventional PCR and qPCR cannot detect both JEECV and AngHV-1 at the same time as they should perform in separate reactions. Thus, the duplex qPCR developed herein is a sensitive and specific diagnostic tool for the simultaneous detection of JEECV and AngHV-1 and has subsequent applications in the health management of eels.

Abstract 979: Development of a methylation-based classifier to identify pancreatic adenocarcinoma subtype

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) demonstrates variable prognosis and response to first-line FOLFIRINOX therapy depending on molecular subtype; patients with classical tumors showed a greater response compared to basal-like tumors. Identifying molecular subtype may inform treatment approaches, but current subtyping schemas rely on tumor biopsies which are invasive and can be technically challenging. Blood based epigenetic assays are commercially available, and identification of subtype-associated epigenetic modifications may provide a path toward blood-based PDAC molecular subtyping. We analyzed methylation levels and developed a subtype classifier using methylation data on PDAC tumors. Using this classifier, we then developed a methylation-specific qPCR assay to subtype PDAC tumors by analyzing methylation at particular sites in tumor DNA which may now be further adapted to more facile liquid biopsy approaches. Methods: Using methylation data (n=150) from TCGA PAAD, we applied a random forest (RF) model followed by a k-Top Scoring Pairs (k-TSP) algorithm to develop a methylation site-based classifier that allows prediction of subtype by comparing methylation levels between paired sites. The classifier was validated on two independent datasets, including ICGC (n=82) and patient-derived xenografts (PDXs) (n=21). We designed methylation-specific qPCR primers for these sites and validated their specificity with fully methylated or fully unmethylated DNA. Using genomic DNA extracted from 10 PDAC PDXs of known molecular subtype, we obtained methylation levels at sites identified by the classifier via a SYBR Green-based qPCR assay. We then used our classifier to predict subtype of the PDX tumors. Results: We validated our RF-kTSP methylation subtype classifier on two independent cohorts and predicted subtype using 20 methylation sites. In ICGC, we found balanced accuracy=0.94, AUROC=0.98, AU-PR=0.89. In PDX PDAC tumors, we found balanced accuracy=1, AUROC=1, AU-PR=1. We designed 14/20 primers for qPCR and confirmed specificity for methylated DNA. Using 14/20 methylation sites, we achieved 80% correct subtype prediction of 10 PDX tumors (5/5 basal-like, 3/5 classical). Conclusion: We have developed a methylation-based classifier that accurately and replicably predicts PDAC subtype. We continue to develop a qPCR assay for PDAC subtype that so far has 80% accuracy using 14/20 probes. We are completing validation of the remaining 6 primers and correlating methylation levels at each site with those obtained from EPIC array methylation analysis. The long-term goal is to apply our complete set of primers in a digital droplet PCR assay that will be amenable for subtyping using circulating tumor DNA (ctDNA). Citation Format: Zachary Schrank, Des Weighill, Hannah Thel, Hannah Trembath, Ashley Morrison, Jen Jen Yeh. Development of a methylation-based classifier to identify pancreatic adenocarcinoma subtype [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 979.

Differential Expression Patterns of Indoleamine 2,3-Dioxygenase 1 and Other Tryptophan and Arginine Catabolic Pathway Genes in Dengue Correlate with Clinical Severity-Pilot Study Results.

The kynurenine pathway of tryptophan catabolism can modulate inflammatory responses inducing immunotolerance or immunosuppressive effects. Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in this pathway. Early aberrant inflammation is implicated in severe dengue, and herein we investigate and characterize the expression of IDO pathway genes in severe dengue patients. We use a SyBR green-based qPCR to evaluate the leukocyte expression levels of IDO1, IDO2, AhR, TGF-β, ARG1, IFNγ, and IFNα in a dengue patient cohort (n = 51). Twenty-two cases were identified as severe dengue using the WHO case classification (2009) criteria. Principal component analysis (PCA) was employed to examine the relationships of gene expression profiles with disease severity and laboratory markers of clinical severity. We find that two principal components describe most of the variance (65.3%) in the expression patterns of the cohort. Reduced expression of IDO1, TGF-β, and AhR, represented by low Component 2 scores, was significantly associated with disease severity, thrombocytopenia, and leukopenia. Higher expression levels of IDO2, IFNγ, and IFNα positively correlated with Component 1 scores, and were significantly associated with elevated ALT (p = 0.018) and AST (p = 0.017) enzymes. Our results suggest that profiling the baseline expression patterns of the IDO pathway genes may aid in the identification of dengue patients most at risk of severe disease.

Quantification of chlamydiae in the endangered Houston toad (Anaxyrus houstonensis).

Two primer set/probe combinations targeting variable regions on the 23S rRNA gene were designed to detect and quantify chlamydiae in DNA extracted from brain swabs of the endangered Houston toad (Anaxyrus houstonensis) using SYBRGreen- and Taqman-based quantitative polymerase chain reaction (qPCR). Prevalence and abundance values for samples were generally different between SYBRGreen- and Taqman-based detection methods, with higher specificity observed for Taqman-based detection. Of the 314 samples analyzed, initial screening with SYBRGreen-based qPCR retrieved 138 positive samples, of which 52 were confirmed by Taqman-based analyses as chlamydiae. All of these samples were subsequently identified as C. pneumoniae by specific qPCR and confirmed by comparative sequence analyses of 23S rRNA gene amplicons. These results demonstrate the usefulness of our developed qPCR methods to screen for and verify prevalence of chlamydiae in DNA of brains swabs, and ultimately specifically identify and quantify chlamydiae and specifically C. pneumoniae in these samples.

Two-Tailed RT-qPCR for the Quantification of A-to-I-Edited microRNA Isoforms.

MicroRNAs are short non-coding RNAs with important functions in the regulation of gene expression in healthy and diseased tissues. To optimally utilize the biological and clinical information that is contained in microRNA expression levels, tools for their accurate and cost-effective quantification are needed. While the standard method, qPCR, allows for quick and cheap microRNA quantification, specificity is limited due to the short lengths of microRNAs and the high similarity between closely related microRNA family members. A-to-I editing can further diversify the microRNA pool by altering individual nucleotides. There is currently a lack of protocols for the accurate quantification of A-to-I-edited microRNA isoforms using qPCR. Here, we describe a protocol to quantify microRNA editing isoforms using two-tailed RT-qPCR, with either SYBR Green or hydrolysis probes. The user will perform reverse transcription of RNA samples, generate standard curves, and quantify the resulting cDNA in the following qPCR step. We also give guidelines for primer design and for the evaluation of assays using synthetic oligonucleotides. These tools are expected to be transferable to any A-to-I-edited microRNA and its isoforms. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Two-tailed reverse transcription of A-to-I-edited microRNAs Basic Protocol 2: SYBR Green-based qPCR for A-to-I-edited microRNAs Alternate Protocol: Hydrolysis probe-based qPCR for A-to-I-edited microRNAs Support Protocol: Preparation of standard curves using synthetic RNA oligonucleotides.

Evaluation of DNA extraction protocols and real-time PCR-based methods for efficient investigation of pig traces in foods

Industrially processed foods are composed of a complex mixture of molecules combined under specific chemical and physical conditions. Besides their native interactions, most of the ingredients included in processed foods are highly transformed through extreme heat variations, grinding, freezing, pH, and pressure fluctuations in order to reach the desired final product. Due to their complex structure and high level of degradation, processed foods are difficult to analyse. Undeclared components are often detected in processed foods, and accurate diagnostic testing is required to protect those with health, cultural, and religious restrictions. Molecular biology techniques involving PCR are most frequently used for determining the authenticity of foods containing derivatives of living organisms. In the present work, we investigated four different DNA extraction protocols of three commercial kits, two different quantitative PCR (qPCR) techniques, and six different primer pairs. We analysed 96 extracts (12 samples from each of the eight products) by SYBR Green-based qPCR using the two most specific and sensitive primer pairs, and compared these results to those obtained with standard commercial kits that use dual dye-labelled probes. Adopting high-efficiency DNA extraction protocols, our findings highlighted the importance of targeting several small regions of the mitochondrial genome to effectively detect small traces of porcine products, and reduce the risk of false-negative results. Adopting these will ensure that consumers can make accurate and informed choices.

Development and Validation of SYBR Green-Based qPCR Technique of Detection and Quantification of <i>Salmonella enterica</i>

Salmonella enterica serovars is a leading cause of human gastroenteritis, and the incidence of salmonellosis is constantly increasing, causing millions of infections and many deaths annually. The detection of the pathogen in optimal terms is an essential factor for reducing the impact on the human body. In this work, SYBR Green I-based qPCR method of detection and quantification of Salmonella enterica was developed and validated. For detection of Salmonella enterica subsp. enterica, two pairs of primers were designed using publically available Primer-BLAST software. Primer efficiency was calculated by establishing a standard curve. The specificity, sensitivity, accuracy, and precision of PCR results were tested. Both primer pairs showed an acceptable performance, proving the developed techniques were sensitive, reliable and precise. The validated qPCR technology has a good potential to replace the traditional culture method in microbial diagnosis.

A sensitive and specific SYBR Green-based qPCR assay for detecting scale drop disease virus (SDDV) in Asian sea bass.

Scale drop disease virus (SDDV) is a megalocytivirus known to cause disease in Asian sea bass in Southeast Asia. To support SDDV diagnosis and surveillance, we report on a sensitive and specific SYBR Green qPCR assay. The qPCR primers were designed to target a 135 bp fragment of the SDDV ATPase gene. The optimized SDDV qPCR assay reliably detected 2 copies of a plasmid dsDNA control and did not cross-amplify DNA to any of 12 viral or bacterial pathogens commonly found in aquatic animals. When assessed with 86 field samples, the assay detected SDDV in DNA extracted from each of 34 scale drop disease-affected fish collected from 5 affected farms. The qPCR also detected SDDV in DNA from 30 of 52 overtly healthy fish collected from 9 farms where SDDV had not been detected previously, using a semi-nested conventional PCR. The higher sensitivity of our SDDV qPCR assay can thus be useful in detecting fish with subclinical/chronic infections. However, the qPCR showed that SDDV DNA loads varied from 8.0 × 102 to 6.8 × 104 viral DNA copies per 200 ng DNA template among the 8 organ tissue types sampled from 3 diseased fish. In circumstances requiring SDDV to be detected unequivocally in subclinical carriers with lower-level infection, qPCR testing of more than one type of tissue is advisable.

Validation of qPCR assays for the detection of citrus canker

Citrus canker, a serious bacterial disease affecting the citrus industry worldwide, is caused by Xanthomonas citri subsp. citri (Xcc) pathotypes A, A* and Aw, and to a lesser extent by X. fuscans subsp. aurantifolii (Xfa). The recent citrus canker outbreak in Australia has emphasised the need to re-evaluate the efficiency of molecular assays used for detecting citrus canker bacteria. Two published probe-based qPCR assays targeting the pth and lrp genes were tested for Xcc, whereas a SYBR Greenbased qPCR assay was tested for Xfa. The Xcc pth gene and Xfa qPCR assays were shown to be specific towards all pathotypes of Xcc and Xfa, respectively. The detection limit for both assays were 1 pg of genomic DNA or 103 CFU in bacteria-spiked leaf sample. The Xcc lrp gene qPCR assay was able to discriminate Xcc pathotypes with a detection limit of 1 ng of genomic DNA or 106 CFU in bacteriaspiked leaf sample, but this assay showed cross-reaction with Xfa. To allow rapid high-throughput detection of all Xcc pathotypes, a duplex probe-based qPCR assay was developed by incorporating COX primers as an internal control for plant DNA into the pth gene qPCR assay.

Comparison of quantitative methods based on SYBR Green real-time qPCR to estimate pork meat adulteration in processed beef products

Quantitative real-time PCR (qPCR) is a modern technique that has been widely used for the detection of species used in meat products. For obtaining accurate and reliable qPCR results, we assessed two common DNA quantification methods for isolated DNA and five quantification approaches for qPCR products. DNA dilution based on spectrofluorometric results showed better qPCR results than those based on spectrophotometry in terms of linear correlation, amplification efficiency, and linear dynamic range. Binary pork-beef mixtures were used to construct standard curves of SYBR Green-based qPCR products using five quantification approaches, and they were validated and compared using in-house pork models. 18S rRNA gene normalization methods showed better trueness (−11.79% to −6.73%) than that of methods using absolute and relative standard curves (−28.52% to −18.64%) in a model burger. These normalized reference methods successfully estimated the quantities of pork meat in the range of 100%–0.01% in commercial beef products.

New set of PCR primers for SYBR green-based qPCR detection of IMNV in India

Emerging and existing diseases are the major havoc to the shrimp aquaculture industry. Outbreaks of viral epidemics severely hinder the sustainable farming system with significant economic losses worldwide. The Pacific white shrimp, Penaeus vannamei farming was seriously affected by infectious myonecrosis caused by a double-stranded RNA virus, infectious myonecrosis virus (IMNV). Early and rapid diagnostics is the priority on considering the efficient management and prevention measures. So, the present study utilized the accurate, rapid and specific detection capabilities of real-time PCR on SYBR Green platform to diagnose and quantify the viral load from the infected tissues by designing an efficient PCR primer set. The developed PCR could detect the virus at 98% efficiency with 10 viral copy number as the limit of detection. The standard curve analysis and amplification arithmetics have shown that it can even detect even less than 10 copy numbers of virus in a sample. The standard curve of the assay has shown R2 value of 0.98 and slope of −3.3834 without any significant variations in inter- and intra- assays. The validated PCR primer pairs and developed SYBR green-based real-time PCR is highly specific, equally sensitive and comparatively economic than the existing TaqMan probe-based PCR for detection of IMNV.

CK19, CK20, EGFR and HER2 status of circulating tumor cells in patients with breast cancer.

The major cause of death in breast cancer patients is metastasis. Various biomarkers have been used for the early detection of circulating tumor cells in the peripheral blood of breast cancer patients. The aims of the current study were to analyze circulating tumor cells in the blood of breast cancer patients by investigating EGFR, CK19, CK20 and HER2 expression profiles and to evaluate their prognostic importance. CK19, CK20 and EGFR gene expression profiles were evaluated in the blood samples of 84 female patients with primary invasive ductal breast cancer and 20 healthy female volunteers using SYBR green-based real-time qPCR assays. HER2 expression analyses were conducted in 46 patients who had an HER2-positive primary tumor and in 30 healthy women to determine the cutoff level of positivity. The positive rates of CK20, EGFR, CK19 and HER2 mRNA expression in the peripheral blood were 28.57% (24/84), 20.23% (17/84), 5.95% (5/84) and 2.17% (1/46), respectively. The high positive ratio of CK20 mRNA expression in the peripheral blood of breast cancer was identified for the first time in the current study. Significant differences were identified in CK20 expression status and several clinical parameters related with aggressiveness of tumors using a binary logistic regression analysis. Higher CK20-positive levels were observed in patients who had lymph node metastasis and advanced-grade primary tumors, which were estrogen receptor-negative. We have demonstrated that CK20 may be a novel biomarker that is useful to identify circulating tumor cells and predict breast cancer progression. The results suggest that the investigation of CK20 mRNA with other biomarkers in the peripheral blood of breast cancer patients may be useful to monitor the presence of disseminated tumor cells in the blood circulation and to predict the prognosis of breast cancer.

Development of a SYBR green-based real-time quantitative PCR assay to detect PCV3 in pigs

Porcine circovirus 3 (PCV3) has been reported in cases of porcine dermatitis and nephropathy syndrome, reproductive failure, cardiac and multi-systemic inflammation. A SYBR green-based real-time quantitative PCR (qPCR) assay was established in this study to detect PCV3 in 203 clinical samples from suckling piglets affected by congenital tremors in China. The limit of detection (LOD) of PCV3 was 1.73×104 copies/μL for gel electrophoresis and 1.73×102 copies/μL for SYBR green-based real-time qPCR. The melt curve analysis showed a single melt peak at 82.5°C.The intra-assay coefficient of variation was up to 1.83% and the inter-assay coefficient of variation was up to 2.27%. The PCV3 positive detection rate of 203 clinical samples for the SYBR green-based real-time qPCR and the conventional PCR was 86.70% (176/203) and 26.60% (54/203), respectively. Each tissue detected in the SYBR green-based real-time qPCR showed a higher positive rate than that detected in the conventional PCR. These results indicated that the SYBR green-based real-time qPCR assay is a powerful method with high sensitivity, specificity and reproducibility for epidemiological investigations of PCV3.

The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis

BackgroundLeishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area.MethodsDNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed.ResultsThe qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the Cq values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of Cq values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples.ConclusionsA new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment.

Library Quantification Using SYBR Green-Quantitative Polymerase Chain Reaction (qPCR).

To quantify complex DNA libraries, Kapa Biosystems engineered a DNA polymerase specifically for SYBR Green-based qPCR, enabling efficient amplification of targets such as GC-rich DNAs that present a challenge to wild-type DNA polymerases. Kapa Library Quantification Kits contain this engineered polymerase to ensure robust amplification of longer fragments, across a broad range of GC content.

MicroRNA-27a-3p regulates epithelial to mesenchymal transition via targeting YAP1 in oral squamous cell carcinoma cells.

MicroRNAs (miRNAs) are small non-coding RNAs frequently dysregulated in human malignancies. Here, we profiled isolated cells from freshly resected tumors from oral squamous cell carcinoma (OSCC) patients and OSCC cell lines using a SYBR Green-based qPCR miRNA array to identify the expression change of the miRNAs. Based on the microarray data and clincopathological factor analysis of 50 OSCC patients related to these miRNAs, miR-27a-3p was selected as a putative miRNA which might play important role in OSCC progression. By bioinformatics analysis and dual-luciferase reporter assay, we found that YAP1 (Yes-associated protein-1) was a direct target gene of miR-27a-3p. Intriguingly, increased expression of miR-27a-3p could significantly decrease the expression level of YAP1 as well as several epithelial to mesenchymal transition (EMT)-related molecules in OSCC cell lines, including Twist and Snail. Then, follow-up studies revealed that miR-27a-3p expression was able to downregulate the EMT-related molecules effectively, which might be involved in the regulation of Sox2 via the YAP1-OCT4-Sox2 signaling axis. In summary, this study found that miR-27a-3p could inhibit the YAP1 directly by post-transcriptionally silencing and potentially suppress EMTprocess, suggesting that miR‑27a-3p might play pivotal roles in effectively manipulating the invasion and metastasis in oral squamous cell carcinoma cells through the EMT inhibition.

P09.44 A poct-adaptable test for the simultaneous identification ofn. gonorrhoeaeand its ciprofloxacin susceptibility status

Introduction Every year, Neisseria gonorrhoeae (Ng) causes 106 million new gonorrhoea infections worldwide and has recently joined the fast-expanding group of multi-drug resistant superbugs. Treatment is often empirical/delayed as culture-based antibiotic susceptibility tests in current use can take several days. A Point-of-Care Test (POCT) for antimicrobial susceptibility would change this; for example, no longer recommended antibiotics such as ciprofloxacin could be used in certain regions where high percentages of isolates remain susceptible. The objective of the present study was to develop and validate a portfolio of diagnostic primer pairs for application in a Nucleic Acid Amplification-based POCT for the concurrent diagnosis of Ng and ciprofloxacin susceptibility. Methods A bioinformatics analysis of >30,000 bacterial genomes identified 9 unique signature sequences specific to Ng FA1090 that were used as targets in a SYBR green-based qPCR assay. 9 diagnostic primer pairs were evaluated for specificity and sensitivity on 271 Ng and non-gonococcal isolates. Two primer pairs targeting ciprofloxacin resistance-conferring single nucleotide polymorphisms in gyrA were tested on 200 resistant and susceptible Ng isolates. qPCR was performed by an Applied Biosystems StepOnePLus TM qPCR system. Results Out of the 9 diagnostic primer pairs tested for Ng identification, one probe failed to detect 28 positive samples of Ng out of a total of 234 isolates. Another primer pair, which amplifies the DR9 repeat region used in the COBAS4800 was not able to detect one Ng isolate collected from Hong Kong. The remaining 7 primer pairs showed 100% specificity in terms of Ng detection and were highly sensitive in detecting Ng DNA in concentrations as low as 0.00001 ng/ul. A multiplexed assay using ciprofloxacin susceptibility-determining primer pairs distinctly differentiated between resistant and susceptible isolates based on melt curve analysis. Conclusion A POCT-adaptable assay has been developed for the simultaneous identification of N. gonorrhoeae and its ciprofloxacin susceptibility status. Disclosure of interest statement The present work was supported by Grand Challenges Canada ((#S5 398). No grants were received from any company in the development of this study.