TaqMan Real-time Reverse Transcription-polymerase Chain Reaction Research Articles

Chemical profiling and unraveling of anti-COVID-19 biomarkers of red sage (Lantana camara L.) cultivars using UPLC-MS/MS coupled to chemometric analysis, in vitro study and molecular docking

Ethnopharmacological relevanceRed sage (Lantana camara L.) (Verbenaceae) is a widely spread plant that was traditionally used in Brazil, India, Kenya, Thailand, Mexico, Nigeria, Australia and Southeast Asia for treating several ailments including rheumatism and leprosy. Despite its historical role in relieving respiratory diseases, limited studies progressed to the plant’s probable inhibition to respiratory viruses especially after the striking spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Aim of the studyThis study aimed to investigate the inhibitory activity of different L. camara cultivars to SARS-CoV-2, that was not previously inspected, and clarify their mechanisms of action in the metabolomics viewpoint, and to determine the biomarkers that are related to such activity using UPLC-MS/MS coupled to in vitro-studies and chemometric analysis. Materials and methodsChemical profiling of different cultivars was accomplished via UPLC-MS/MS. Principle component analysis (PCA) and orthogonal projection to latent structures (OPLS) models were built using SIMCA® (multivariate data analysis software). Cytotoxicity and COVID-19 inhibitory activity testing were done followed by TaqMan Real-time RT-PCR (Reverse transcription polymerase chain reaction) assay that aimed to study extracts' effects on RNA-dependent RNA polymerase (RdRp) and E-genes expression levels. Detected biomarkers from OPLS analysis were docked into potential targets pockets to investigate their possible interaction patterns using Schrodinger® suite. ResultsUPLC-MS/MS analysis of different cultivars yielded 47 metabolites, most of them are triterpenoids and flavonoids. PCA plots revealed that inter-cultivar factor has no pronounced effect on the chemical profiles of extracts except for L. camara, cultivar Drap d'or flowers and leaves extracts as well as for L. camara cv Chelsea gem leaves extract. Among the tested extracts, flowers and leaves extracts of L. camara cv Chelsea gem, flowers extracts of L. camara cv Spreading sunset and L. camara cv Drap d'or showed the highest selectivity indices scoring 12.3, 10.1, 8.6 and 7.8, respectively, indicating their relative high safety and efficacy. Leaves and flowers extracts of L. camara cv Chelsea gem, flowers extracts of L. camara cv Spreading sunset and L. camara cv Drap d'or were the most promising inhibitors to viral plaques exhibiting IC50 values of 3.18, 3.67, 4.18 and 5.01 μg/mL, respectively. This was incremented by OPLS analysis that related their promising COVID-19 inhibitory activities to the presence of twelve biomarkers. Inhibiting the expression of RdRp gene is the major mechanism behind the antiviral activity of most extracts at almost all concentration levels. Molecular docking of the active biomarkers against RdRp revealed that isoverbascoside, luteolin-7,4'-O-diglucoside, camarolic acid and lantoic acid exhibited higher docking scores of −11.378, −10.64, −6.72 and −6.07 kcal/mol, respectively, when compared to remdesivir (−5.75 kcal/mol), thus these four compounds can serve as promising anti-COVID-19 candidates. ConclusionFlowers and leaves extracts of four L. camara cultivars were recognized as rich sources of phytoconstituents possessing anti-COVID-19 activity. Combination of UPLC-MS/MS and chemometrics is a promising approach to detect chemical composition differences among the cultivars and correlate them to COVID-19 inhibitory activities allowing to pinpoint possible biomarkers. Further in-vitro and in-vivo studies are required to verify their activity.

Comparison and evaluation of conventional RT-PCR, SYBR green I and TaqMan real-time RT-PCR assays for the detection of porcine epidemic diarrhea virus

Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is a highly contagious intestinal disease, resulting in substantial economic losses to the swine industry worldwide. In this study, three assays, namely a conventional reverse transcription-polymerase chain reaction (RT-PCR), a SYBR Green I real-time RT-PCR and a TaqMan real-time RT-PCR targeting the highly conserved M gene of PEDV, were developed and evaluated. Then, the analytical specificity, sensitivity and reproducibility of these assays were determined and compared. The TaqMan real-time RT-PCR was 100-fold and 10,000-fold more sensitive than that of the SYBR Green I real-time RT-PCR and the conventional RT-PCR, respectively. The analytical sensitivity of TaqMan real-time RT-PCR was 10 copies/μl of target gene and no cross amplification with other viruses tested was observed. With the features of high specificity, sensitivity, and reproducibility, the TaqMan real-time RT-PCR established in this study could be a useful tool for clinical diagnosis, epidemiological surveys and outbreak investigations of PED.

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses

Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay.

A TaqMan real-time RT-PCR assay for detection of Groundnut ringspot virus.

Groundnut ringspot virus (GRSV) is a tospovirus species transmitted in a circulative propagative manner by several thrips species. Traditionally, GRSV is detected by enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR), so this paper describes a rapid diagnostic system for the reliable detection of GRSV in plants and individual thrips by real-time RT-PCR. Watermelon GRSV infected leaves and fruits, and individual thrips (Frankliniella schultzei) collected from flowers on infected watermelons were used in the test. We designed a GRSV TaqMan assay to efficiently detect GRSV in all samples. The method had high specificity and could distinguish GRSV from the other tospovirus species, like Tomato spotted wilt virus (TSWV), Tomato chlorotic spot virus (TCSV) and Zucchini lethal chlorosis virus (ZLCV). The test will help us to collect epidemiological data for GRSV in plants and thrips worldwide.

Development of TaqMan real-time reverse transcription-polymerase chain reaction for the detection and quantitation of porcine kobuvirus

Porcine kobuvirus (PKV) is a newly emerging virus that has been detected in diarrheic pigs. Presently, reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated amplification are the only methods that can be used to detect PKV. To develop a TaqMan real-time RT-PCR for the rapid detection and quantitation of PKV nucleic acid in fecal samples, a pair of primers and a probe were designed to amplify the conserved 3D region of the PKV genome. After optimization, the TaqMan real-time RT-PCR was highly specific and ∼1000 times more sensitive than conventional RT-PCR, and the detection limit was as low as 30 DNA copies. Among the 148 intestinal samples from piglets with diarrhea, 136 and 118 were positive based on the TaqMan and conventional RT-PCR methods, respectively, indicating that the TaqMan RT-PCR was more sensitive than conventional RT-PCR, and the total concordance of the two methods was approximately 87.84%. Thus, the TaqMan real-time RT-PCR should be a useful tool for the early detection and quantitation of PKV.

Expression profile of six stress-related genes and productive performances of fast and slow growing broiler strains reared under heat stress conditions

High temperature is one of the prominent environmental factors causing economic losses to the poultry industry as it negatively affects growth and production performance in broiler chickens. We used One Step TaqMan real time RT-PCR (reverse transcription polymerase chain reaction) technology to study the effects of chronic heat stress on the expression of genes codifying for the antioxidative enzymes superoxide dismutase (SOD), and catalase (CAT), as well as for heat shock protein (HSP) 70, HSP90, glucocorticoid receptor (NR3C1), and caspase 6 (CASP6) in the liver of two different broiler genetic strains: Red JA Cou Nu Hubbard (CN) and Ross 508 Aviagen (RO). CN is a naked neck slow growing broiler intended for the free range and/or organic markets, whereas RO is selected for fast growing. We also analysed the effect of chronic heat stress on productive performances, and plasma corticosterone levels as well as the association between transcriptomic response and specific SNPs (single nucleotide polymorphisms) in each genetic strain of broiler chickens. RO and CN broilers, 4weeks of age, were maintained for 4weeks at either 34°C or 22°C. The results demonstrated that there was a genotype and a temperature main effect on the broilers' growth from the 4th to the 8th week of age, but the interaction effect between genotype and temperature resulted not statistically significant. By considering the genotype effect, fast growing broilers (RO) grew more than the slow growing ones (CN), whereas by considering the temperature effect, broilers in unheated conditions grew more than the heat stressed ones. Corticosterone levels increased significantly in the blood of heat stressed broilers, due to the activation of the HPA (hypothalamic–pituitary–adrenocortical axis). Carcass yield at slaughter was of similar values in the 4 cohorts (genotype/temperature combinations or treatment groups), ranging from 86.5 to 88.6%, whereas carcass weight was negatively influenced by heat stress in both broiler strains. Heat stress affected gene expression by downregulating CASP6 and upregulating CAT transcript levels. HSPs, SOD and NR3C1 mRNA levels remained unaffected by heat stress. The differences found in the mRNA copies of CASP6 gene could be partly explained by SNPs.

Detection of measles virus RNA on SYBR green real‐time reverse transcription–polymerase chain reaction

As the coverage rate of the measles vaccine increases, not all patients present the typical symptoms of measles after exposure to the measles virus (MV). The virus loads in clinical specimens from patients with vaccine-modified non-typical measles are expected to be low compared with those of primary MV infection. A rapid and sensitive laboratory procedure is required for diagnosis of measles. SYBR Green (TaKaRa) and TaqMan (ABI) real-time reverse transcription-polymerase chain reaction (RT-PCR) assays were developed to detect MV-RNA. For the real-time RT-PCR, primer sets were designed from a region of the MV H gene of the Edmonston strain (genotype A). A TaqMan probe specific for the H gene of genotype D MV was used. The minimum detectable level of MV-RNA in the SYBR Green and TaqMan real-time RT-PCR assays was evaluated using synthetic MV-RNA. The sensitivity of real-time RT-PCR was compared with that of nested RT-PCR and the virus isolation method using throat swabs and peripheral blood samples from patients with measles. The minimum detectable level of RNA was 10 and 10(2) copies for SYBR Green RT-PCR and TaqMan RT-PCR, respectively. Ten-10(6) copies of standard RNA were linearly detected on SYBR Green RT-PCR. The sensitivity of SYBR Green RT-PCR was equal to that of nested RT-PCR. MV-RNA was detected in virus isolation-negative throat swabs on SYBR Green RT-PCR. SYBR Green RT-PCR is a highly sensitive, rapid, and useful diagnostic procedure for the detection of MV.

Real time TaqMan RT-PCR assay for the detection of Cucumber green mottle mosaic virus

A real time reverse-transcription polymerase chain reaction (RT-PCR) was developed for efficient detection of Cucumber green mottle mosaic virus (CGMMV). The method was designed to use a duo-primer system with a TaqMan probe targeting the conserved sequence in 3′ noncoding region (NCR) of CGMMV to detect isolates of this virus collected in China. The sensitivity of the real time RT-PCR assay was 0.13 pg of total RNA or 50 molecules of RNA transcripts. This level of sensitivity indicated that the one step real time RT-PCR developed in the present study could be used for routine testing assays. The real time RT-PCR method could assist in the implementation of quarantine measures for prevention and control of the disease caused by CGMMV.

Detection and quantification of human and bovine noroviruses by a TaqMan RT-PCR assay with a control for inhibition

Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and calves. Reverse transcription-polymerase chain reaction (RT-PCR) has become the "gold standard" for detection of noroviruses in faecal and environmental samples. However, false negative results due to co-concentration of RT-PCR inhibitors are a continuous concern. A TaqMan real-time RT-PCR assay making use of a foreign internal RNA control and a RNA standard was developed. Very interestingly, this method is capable of detecting human noroviruses belonging to genogroups I and II, and bovine noroviruses belonging to genogroup III. Inhibitors were removed efficiently by 1/10 dilution of the sample or addition of bovine serum albumin to the RT-PCR mix. This assay was validated with human and bovine stool samples previously tested for norovirus by conventional RT-PCR. The ability to detect norovirus in stool samples that were negative by conventional RT-PCR assay demonstrate the higher sensitivity of the TaqMan assay compared to the conventional RT-PCR assay. This real-time RT-PCR assay allows the detection of both human and bovine noroviruses, avoids false negative results and is able to quantify the level of norovirus contamination.

Development of a one-step immunocapture real-time TaqMan RT-PCR assay for the broad spectrum detection of Pepino mosaic virus

A real-time reverse-transcription polymerase chain reaction (RT-PCR) was developed for efficient detection of genetically diverse Pepino mosaic virus (PepMV) isolates. The novel detection system was designed to use a duo-primer system targeting the conserved region in the triple gene block 2 (TGB2) gene with a single conserved TaqMan™ probe to broaden its reaction to cover all available PepMV strains. This duo-primer real-time RT-PCR assay was evaluated against US1, US2, Ch1, Ch2 and 25 field isolates collected from six major commercial tomato greenhouse facilities in U.S. and Canada in 2006. Under optimum reaction conditions, sensitivity of the detection was as low as 100 fg of purified viral RNA. This assay was also evaluated for its efficiency in detecting PepMV in various levels of contaminated seed samples. Using immunocapture sample preparation, real-time RT-PCR was able to detect PepMV in one infested seed in 1000. This level of sensitivity indicated that the one-step immunocapture duo-primer TaqMan™ real-time RT-PCR developed in the present study could be used for routine seed health assays.

A Fully Automated Procedure for the High-Throughput Detection of Avian Influenza Virus by Real-Time Reverse Transcription–Polymerase Chain Reaction

The recent spread of highly pathogenic H5N1 avian influenza (AI) has made it important to develop highly sensitive diagnostic systems for the rapid detection of AI genome and the differentiation of H5N1 variants in a high number of samples. In the present paper, we describe a high-throughput procedure that combines automated extraction, amplification, and detection of AI RNA, by an already described TaqMan real-time reverse transcription-polymerase chain reaction (RRT-PCR) assay targeted at the matrix (M) protein gene of AI virus (AIV). The method was tested in cloacal and tracheal swabs, the most common type of samples used in AI surveillance, as well as in tissue and fecal samples. A robotic system (QIAGEN Biosprint 96) extracted RNA and set up reactions for RRT-PCR in a 96-well format. The recovery of the extracted RNA was as efficient as that of a manual RNA extraction kit, and the sensitivity of the detection system was as high as with previously described nonautomated methods. A system with a basic configuration (one extraction robot plus two real-time 96-well thermocyclers) operated by two persons could account for about 360 samples in 5 hr. Further characterization of AI RNA-positive samples with a TaqMan RRT-PCR specific for H5 (also described here) and/or N1 was possible within 2 hr more. As this work shows, the system can analyze up to 1400 samples per working day by using two nucleic acid extraction robots and a 384-well-format thermocycler.

Application of real-time polymerase chain reaction for the quantitation of interleukin-1β mRNA upregulation in brain ischemic tolerance

Differential gene expression plays an important role in normal development and pathophysiological conditions. The accurate quantitation of mRNA expression is critical to assess the differential gene expression. While a number of techniques, such as Northern analysis, (semi-)quantitative reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization, are available to measure the levels of mRNA expression, certain limitations exist, including the insensitive and inaccurate quantitation of mRNA expressed at low abundance. In the present study, we describe the application of a recently developed TaqMan real-time quantitative RT-PCR for the detection of interleukin-1β (IL-1β) mRNA expression in rat cortical tissue after a short duration of ischemia (i.e., ischemic preconditioning). The principle of the TaqMan real-time detection is based on the fluorogenic 5′ nuclease assay that allows simple and rapid quantitation of a target sequence during the extension phase of PCR amplification. Using a cloned plasmid DNA as a standard and normalizing RNA samples with a housekeeping gene for the TaqMan real-time PCR, we detected the significant induction in absolute copy numbers of IL-1β mRNA in the ipsilateral cortex after preconditioning, suggesting a potential role of this inflammatory cytokine in ischemic brain tolerance. Themes: Disorders of the nervous system Topics: Ischemia