Single RT-qPCR Research Articles

Establishment and Application of a Triplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assay for Differentiation of PEDV, TGEV and PKV.

The pathogens responsible for porcine viral diarrhea are diverse, causing significant economic losses to the pig industry. PEDV and TGEV are well-known pathogens causing diarrheal diseases in pigs, leading to significant economic losses in the breeding industry. In contrast, the newly identified diarrhea virus, PKV, has not garnered as much attention. However, co-infection of PKV with PEDV results in more severe symptoms in piglets, such as acute gastroenteritis, and promotes increased replication of PEDV. Rapid and accurate diagnosis of viral diarrhea is essential for farms to identify pathogens early and mitigate economic losses. This study describes the development of a triplex real-time fluorescent quantitative RT-qPCR technique that can simultaneously detect three RNA viruses associated with porcine viral diarrhea: PEDV, TGEV, and PKV. To establish the triplex RT-qPCR method for the simultaneous detection and identification of the above three diarrhea viruses, conserved regions of the M gene of TGEV, the N gene of PEDV, and the 3D gene of PKV were selected to design specific primers and probes. After optimizing the reaction conditions, the method's specificity, sensitivity, and reproducibility were evaluated. The triplex RT-qPCR method did not show a significant difference in PCR efficiency compared to the single RT-qPCR method. The method is specific to TGEV, PKV, and PEDV, exhibits no cross-reactivity with other pathogens, and demonstrates satisfactory sensitivity and reproducibility; the limit of detection (LOD) of PEDV, TGEV, and PKV is 11.42 copies/μL. Furthermore, the performance of the triplex RT-qPCR assay was compared with the Chinese standard single-assay method for detecting TGEV, PKV, and PEDV, showing complete consistency between the two methods (100% compliant). Subsequently, 1502 clinical diarrhea samples were collected from the Guangxi Zhuang Autonomous Region to investigate the local prevalence of TGEV, PKV, and PEDV and the positive rates were 16.38% (246/1502), 1.46% (22/1502), and 45.14% (678/1502), respectively. Co-infection of PEDV and PKV were most common, with a rate of 12.12% (182/1502). This study presents a valuable method for the rapid and simultaneous identification of PEDV, TGEV, and PKV in clinical animal farming practices, and provides a reassessment of the epidemiology of these diarrhea-causing viral pathogens in the Guangxi Zhuang Autonomous Region.

Denaturing and dNTPs reagents improve SARS-CoV-2 detection via single and multiplex RT-qPCR

Background The COVID-19 pandemic, caused by the SARS-CoV-2, can be effectively managed with diagnostic tools such as RT-qPCR. However, it can produce false-negative results due to viral mutations and RNA secondary structures from the target gene sequence. Methods With High Performance Computing, the complete SARS-CoV-2 genome was obtained from the GenBank/GISAID to generate consensus sequences to design primers/probes for RT-qPCR. ORF8 gene was selected, meanwhile, E and N and RNAse P were according to CDC protocol. Nasopharyngeal swab samples were collected from patients diagnosed with SARS-CoV-2. Total RNA was purified according MagMAX kit, it was used in single, and multiplex RT-qPCR. To avoid templated secondary structures, compensate nucleotide proportions and improve Ct values, a solution composed of tetraethylammonium chloride and dimethyl sulfoxide and other with corresponding to dNTPs proportions in accordance SARS-CoV-2 genome were included. Sensitivity and specificity according to Ct values were determined with the Caret package in R software. Results 126,576 SARS-CoV-2 genomes from January to December 2020 comprised a database. From this, a region near of 5′ ORF8 gene showed three stem-loops was used for primers/FAM-probe. 49 samples were obtained, from them, 22 were positive to gene selected regions. Interestingly, samples from October to November 2020 were positive for all regions, however, in January 2021 different results were observed in ORF8. An improvement in Ct with the adjuvant solutions was determined in all samples with others SARS-CoV-2 primers/probes, for both single and multiplex RT-qPCR. The inclusion of the denaturing solution in single RT-qPCR increased its sensitivity with respect to the commercial method, while in multiplex the opposite was generated. Conclusions Including adjuvant solutions to prevent the formation of RNA secondary structures and the adjustment of the nucleotide ratios of SARS-CoV-2 improved single and multiplex RT-qPCR for viral identification, demonstrating its potential application in health public.

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.

Modelling pooling strategies for SARS-CoV-2 testing in a university setting

Background: Pre-symptomatic and asymptomatic transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are important elements in the coronavirus disease 2019 (COVID-19) pandemic, and there remains a reliance on testing to manage the spread of the disease. In the UK, many universities opened for blended learning for the 2020-2021 academic year, with a mixture of face to face and online teaching. Methods: In this study we present a simulation framework to evaluate the effectiveness of different mass testing strategies within a university setting, across a range of transmission scenarios. Results: The sensitivity of 5x pooled RT-qPCR tests appears to be higher than testing using the lateral flow device with relatively little loss compared to single RT-qPCR tests, and is improved by pooling by social cluster. The range of strategies that we evaluated give comparable results for estimating prevalence. Conclusions: Pooling tests by known social structures, such as student households can substantially improve the cost effectiveness of RT-qPCR tests. We also note that routine recording of quantitative RT-qPCR results would facilitate future modelling studies.

Prognostic Significance of TWIST1, CD24, CD44, and ALDH1 Transcript Quantification in EpCAM-Positive Circulating Tumor Cells from Early Stage Breast Cancer Patients.

(1) Background: The aim of the study was to evaluate the prognostic significance of EMT-associated (TWIST1) and stem-cell (SC) transcript (CD24, CD44, ALDH1) quantification in EpCAM+ circulating tumor cells (CTCs) of early breast cancer patients. (2) Methods: 100 early stage breast cancer patients and 19 healthy donors were enrolled in the study. CD24, CD44, and ALDH1 transcripts of EpCAM+ cells were quantified using a novel highly sensitive and specific quadraplex RT-qPCR, while TWIST1 transcripts were quantified by single RT-qPCR. All patients were followed up for more than 5 years. (3) Results: A significant positive correlation between overexpression of TWIST1 and CD24−/low/CD44high profile was found. Kaplan–Meier analysis revealed that the ER/PR-negative (HR-) patients and those patients with more than 3 positive lymph nodes that overexpressed TWIST1 in EpCAM+ cells had a significant lower DFI (log rank test; p < 0.001, p < 0.001) and OS (log rank test; p = 0.006, p < 0.001). Univariate and multivariate analysis also revealed the prognostic value of TWIST1 overexpression and CD24−/low/CD44high and CD24−/low/ALDH1high profile for both DFI and OS. (4) Conclusions: Detection of TWIST1 overexpression and stem-cell (CD24, CD44, ALDH1) transcripts in EpCAM+ CTCs provides prognostic information in early stage breast cancer patients.

Comparison of three different molecular assays for the detection and molecular characterization of circulating tumor cells in breast cancer

Objective Comparison of three different molecular assays for the detection and molecular characterization of circulating tumor cells in breast cancer. Methods The retrospective study compared three different molecular assays to detect CTC in the peripheral blood of 30 healthy individuals and 71 benign breast disease patients and 83 early and 84 metastatic breast cancer patients.All samples were collected at the outpatient, inpatient and physical examination department of Sichuan Provincial People's Hospital from January 2011 to June 2014. The same cDNAs were analyzed by: singleplex RT–qPCR assay for BCL–2, multiplex RT–qPCR for BCL–2, HER–2, HMAM, and a commercially available molecular assay (AdnaTest BreastCancer) for GA733–2, MUC–1, HER–2. The positive of CTC were compared among healthy individuals and benign breast disease patients and breast cancer patients. Chi square test was used to compare the expression of gene markers among the three groups, and the agreement of Kappa test was used to evaluate the method. Results (1) Detection rates of early breast cancer by single RT–qPCR, Adna kits and multiple RT–qPCR were 13.3%, 16.9% and 18.1%, respectively, and the detection of metastatic breast cancer were 31.0%, 42.9% and 35.7%, respectively. There were significant differences in the positive of CTC by three molecular assays between healthy individuals and benign breast disease patients and early breast cancer patients (The test values were 4.235 and 4.301, 5.367 and 5.474, 5.894 and 6.023 respectively, P 0.05). There were significant differences between metastasis breast cancer patients and healthy individuals and benign breast disease patients and early breast cancer patients (The test values were 8.429, 7.553 and 7.061; 10.24, 9.025 and 8.745; 9.658, 8.417 and 8.201 respectively, P<0.05). (2) In early breast cancer: The concordance between AdnaTest and single RT–qPCR was 79.5% while between AdnaTest and multiplex RT–qPCR was 77.1%.No agreement was found among them (The test values were 1.065 and 1.871, P were 0.371 and 0.258). The concordance between single RT–qPCR and multiplex RT–qPCR was 80.7%.No agreement was found between them (The test values was 2.814, P was 0.156). (3) In patients with overt metastasis: The concordance between AdnaTest and single RT–qPCR was 78.6%(The test values was 10.986). While between AdnaTest and multiplex RT–qPCR was 80.9%(The test values was 9.251). Agreements were found among them (P was 0.002 and 0.005 respectively). The concordance between single and multiplex RT–qPCR was 88.1%(The test values was 12.364). Agreement was found between them (P was 0.001). Conclusions No correlations were found among different molecular methods to detect CTC in the early primary breast cancer, but correlations were found in the metastatic breast cancer, suggesting that different rate of CTC caused by the number of CTC and its heterogeneity should be considered to the clinical diagnosis and treatment of breast cancer while molecular method is used.(Chin J Lab Med, 2015, 38: 666–671) Key words: Breast neoplasms; Neoplastic cells, circulating; Tumor markers, biological; Reverse transcriptase polymerase chain reaction

Rapid detection and high occurrence of porcine rotavirus A, B, and C by RT-qPCR in diagnostic samples

Rotaviruses are important cause of diarrhea in animals, including humans. Currently, rotavirus species A, B, C, E, and H (RVA-RVC, RVE, and RVH) have been identified in pigs. Traditionally, RVA has been considered the primary cause of diarrhea in pigs, and RVB and RVC had been described sporadically in pigs until recently. Qualitative porcine RVA, RVB, and RVC RT-PCR (RT-qPCR) assays were designed and 7508 porcine diarrheic samples, submitted to University of Minnesota, were tested to estimate the percentage of RVA, RVB, and RVC over a period of approximately 2 years (from 2009 to 2011). The individual RVA and RVC RT-qPCR assays were multiplex into a single RT-qPCR while the RVB RT-qPCR assay remained as an individual RT-qPCR. In total, 83% of the samples were positive for RVA, RVB, or RVC. As expected, RVA was detected at the highest overall percentage (62%). However, 33% and 53% of the samples were positive for RVB and RVC, respectively, indicating that both RVB and RVC are also epidemiologically important in the swine population. RVC was most predominant in young pigs (1–20 days of age), while RVA and RVB were most predominant in ≥21 day old pigs. As diagnostic tools, the developed RT-qPCR assays could successfully discriminate among infecting RV species, which could lead to better surveillance and epidemiological studies for ultimately better prevention and control strategies.

A duplex, SYBR Green I-based RT-qPCR assay for the simultaneous detection of Apple chlorotic leaf spot virus and Cherry green ring mottle virus in peach

BackgroundCo-infections of Apple chlorotic leaf spot virus (ACLSV) and Cherry green ring mottle virus (CGRMV) in peach is common in China and have resulted in significant yield reductions. A reliable, sensitive and quantitive method is needed to detect and distinguish between ACLSV and CGRMV in peach.FindingsWe developed a sensitive and specific SYBR Green-I based RT-qPCR for the quantification of ACLSV and CGRMV in different peach tissues, and a duplex RT-qPCR system to detect ACLSV and CGRMV simultaneously. The RT-qPCR method was optimized using standard samples transcribed by the T7 Large Scale RNA Production System in vitro. The peach genes, RNA Polymerase subunit II (RPII) and Ubiquitin 10 (UBQ10), which were used as the internal controls for the quantification assay also showed good expression stability in this system. Single RT-qPCR assays showed that CGRMV in peach accumulates to a higher level than ACLSV. The detection limits of the duplex RT-qPCR assay were 102 and 104 copies for ACLSV and CGRMV, respectively. The sensitivity of the duplex RT-qPCR was as high as RT-qPCR and higher than RT-PCR.ConclusionsThe SYBR Green-I RT-qPCR assay provided a sensitive, specific and reliable method for the detection and quantification of ACLSV and CGRMV in different peach tissues. The duplex RT-qPCR system provided a sensitive and specific method to detect and differentiate between ACLSV and CGRMV in a single sample. This RT-qPCR assay could be a useful tool for the routine diagnosis of these two viruses and for disease epidemiology studies in peach orchards.

Abstract 4150: CK-19, MAGE-A3, HER-2 and TWIST-1 expression by RT-qPCR in CTCs of primary breast cancer patients after positive immune-magnetic selection using anti-EpCAM and anti-MUC1 magnetic beads

Abstract Background: Circulating tumor cells (CTCs) are suggested as potential surrogate markers for minimal residual disease, the precursor of metastatic disease. Furthermore, to be able to disseminate and metastasize, CTCs must be able to perform epithelial-mesenchymal transition (EMT). The aim of our study was to evaluate the sensitivity of RT-qPCR for the detection and characterization of CTCs analyzing the expression of CK-19, MAGE-A3, HER-2 and TWIST-1 as a transcription factor for EMT in blood of early breast cancer patients after positive immunomagnetic selection using a combination of anti-EpCAM and anti-MUC1 magnetic beads. Patients and Methods: Two × 5 ml blood samples were collected before surgery and CTCs were isolated by positive immunomagnetic selection using anti-EpCAM and anti-MUC1 coated magnetic beads (AdnaTest Breast Cancer Select, AdnaGen AG, Langenhagen, Germany). CK-19, MAGE-A3, HER-2 and PBGD transcripts were simultaneously quantified by multiplex RT-qPCR while TWIST-1 transcript by single RT-qPCR in the LightCycler 2.0 (Roche Diagnostics). All primers and probes were designed to match the assay conditions, such as amplicon sizes, and melting temperatures. Before proceeding to patients’ samples, the RT-qPCR assay was evaluated in respect to sensitivity, specificity, and reproducibilty. To ensure that amplifiable material was present in all specimens and to avoid false-negative results, real-time amplification of the reference gene porphobilinogen deaminase (PBGD) was performed for all samples. A total number of 436 samples were first evaluated for RNA quality by PBGD expression and finally 293 samples that were of good RNA quality were further processed. Results: CK-19 was detected in 117/293 (39.9%), HER-2 in 35/293 (12.0%), MAGE-A3 in 25/293 (8.5%), and TWIST-1 in 16/293 (5.5 %) of all samples, respectively. One gene was detected in 135/293 (46.1%) of the patients, two genes in 23/293 (7.8%) of the patients, three genes in 3/293 (1.0%) patients and four genes in 0/293 (0 %) of the patients, respectively. In total, 161/293 (54.9%) patients were found positive for at least one gene expression, while in 132/293 (45.05%) patients we didn't detect CTCs at all. Discussion: Our study has revealed a high sensitivity when using a combination of immunomagnetic separation and RT-qPCR, as well as a remarkable heterogeneity of gene expression for these genes between early breast cancer patients. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4150. doi:10.1158/1538-7445.AM2011-4150

Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Selumetinib (AZD6244, ARRY-142886) is a selective, non-ATP-competitive inhibitor of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR-based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies.