Reverse transcription-PCR Assay Research Articles

Evaluation of interleukin-17 receptor(IL-17RA) gene expression in PBMCs of patients with premature coronaryartery disease.

Present study has been carried out to analyze the IL-17R gene expression in PBMCs of patients with premature coronary artery disease (CAD) in comparison to normal controls. Premature CAD results in disability and lack of quality of life over the years and consequent mortality.Cardiovascular disease (CVD) has global distribution. In 2022, CAD is the leading cause of mortality in the United States and Iran. IL-17 cytokine family plays an important role in promoting inflammation and producing pro-inflammatory cytokines, chemokines, and matrix metalloproteinases. Entirely, 60 subjects were entered into this examination. The case group consisted of 30 subjects with CAD as well as the control group which consisted of 30 healthy persons. The real-time quantitative reverse transcription PCR assay was used to find out, the relative expression (fold) level of IL-17R gene. Our findings indicated that, the relative expression (fold) level of IL-17R gene in the patients group showed an increased level as compared to the control group. The analysis of findingsobtained in this study showed that the patient group is significantly different from the control group regarding the IL-17R mRNA level (fold) (p = 0.035). It has been concluded that IL-17R plays an important role in the pathogenesis of CAD. It follows that superior understanding of IL-17/IL-17R signaling way will be vital for innovating novel therapeutic targets that will facilitate the designing of new drugs for the management of patients (Ref. 40).

Rapid Detection of the Omicron (B.1.1.529) SARS-CoV-2 Variant Using a COVID-19 Diagnostic PCR Assay.

ABSTRACTThe Omicron (B.1.1.529) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the last variant of concern (VOC) identified to date. Compared to whole-genome or gene-specific sequencing methods, reverse-transcription PCR assays may be a simpler approach to study VOCs. We used a point-of-care COVID-19 diagnostic PCR assay to detect the Omicron SARS-CoV-2 variant in the respiratory tract samples of COVID-19 patients who had tested positive for SARS-CoV-2 RNA between April 2021 and January 2022. Sequencing analyses had shown that 87 samples were positive for the Omicron variant and 43 samples were positive for a non-Omicron variant (Delta, 18 samples; Alpha, 13 samples; Gamma, 10 samples; Beta, 1 sample; or Epsilon, 1 sample). According to results by the PCR assay, whose primers anneal a nucleocapsid (N) gene region that comprises the E31/R32/S33 deletion (also termed the del31/33 mutation), we found that N gene target failure/dropout (i.e., a negative/low result) occurred in 86 (98.8%) of 87 Omicron variant-positive samples tested. These results were assessed in relation to those of the spike (S) gene, which expectedly, was detected in all (100%) 130 samples. A total of 43 (100%) of 43 Delta, Alpha, Gamma, Beta, or Epsilon variant-positive samples had a positive result with the N gene. Importantly, in 86 of 87 Omicron variant-positive samples, the del31/33 mutation was detected together with a P13L mutation, which was, instead, detected alone in the Omicron variant-positive sample that had a positive N-gene result.IMPORTANCE Rapid detection of the Omicron SARS-CoV-2 variant in patients’ respiratory tract samples may influence therapeutic choices, because this variant is known to escape from certain monoclonal antibodies. Our findings strengthen the importance of manufacturers’ efforts to improve the existing COVID-19 diagnostic PCR assays and/or to develop novel variant-specific PCR assays. Furthermore, our findings show that only a small fraction of SARS-CoV-2-positive samples may require whole-genome sequencing analysis, which is still crucial to validate PCR assay results. We acknowledge that the emergence of novel variants containing mutations outside the PCR assay target region could, however, allow an assay to work as per specifications without being able to identify a SARS-CoV-2-positive sample as a variant. Future work and more experience in this topic will help to reduce the risk of misidentification of SARS-CoV-2 variants that is unavoidable when using the current PCR assays.

Transcriptome analysis revealed the role of capsular polysaccharides in desiccation tolerance of foodborne Staphylococcus aureus

Staphylococcus aureus (S. aureus) is a momentous factor affecting food safety. It can survive under long-term desiccation stress and contaminate foods that have intermediate to low water activities. However, the specific molecular mechanisms by which it survives and persists under low water activity stress are often overlooked. In this study, transcriptome analysis was applied to investigate the effect of desiccation stress on gene expression of S. aureus RMSA24, a food-borne S. aureus strain that was isolated from a raw milk sample. Results of transcriptome analysis showed that the mRNA levels of genes related to capsular polysaccharides (CPs) synthesis were significantly upregulated after desiccation treatment, which was further confirmed by real-time reverse transcription PCR assays. Furthermore, the results of colony count experiments demonstrated that the survival of CPs mutant was decreased compared with the wild type strain. And the biofilm formation ability of CPs mutant showed no difference compared with that of wild type according to biofilm formation assays. Those results indicated that CPs mutant decrease the resistance to desiccation in S. aureus RMSA24 via a biofilm-independent pathway. This study provides new evidence regarding the mechanisms of desiccation resistance of food-borne S. aureus and contributes to the prevention of food contamination caused by this bacterium.

Detection of Newcastle disease virus and H5-subtype Influenza virus in swiftlet houses by Multiplex reverse transcription PCR assay

The purpose of this study was to investigate the presence of Avian paramyxovirus 1 (APMV-1), also known as Newcastle disease virus (NDV), and Avian influenza type A, especially H5 subtype (AIV-H5) in swiftlet houses and swiftlet nest by multiplex RT-PCR (mRT-PCR). The assay used two specific primer pairs designed to detect the conserved sequence of the F gene of NDV and the HA gene of the AIV-H5, with product sizes of 282 bp and 420 bp, respectively. The mRT-PCR was established with detection limit of 25 copies/reaction for each target virus. The thermal cycle was optimized as follows: cDNA synthesis at 45ºC for 20 min, an initial denaturation at 95ºC for 5 min, followed by 35 cycles of amplification encompassing denaturation at 95ºC for 30 sec, annealing at 58ºC for 30 sec, and extension at 72ºC for 45 sec, ending by a final extension step at 72ºC for 7 min. Eighty-eight field samples including feces and swabs of the nest surface were examined and all samples were confirmed to be negative for these two viruses. The results of this study indicated that the swiftlet nests and the environment of swiftlet houses were not contaminated with NDV or AIV-H5 viruses. Moreover, the established mRT-PCR protocol had good specificity, detection limit, and can apply for routine veterinary diagnosis.

An updated review of SARS-CoV-2 detection methods in the context of a novel coronavirus pandemic.

The World Health Organization has reported approximately 430 million confirmed cases of coronavirus disease 2019 (COVID‐19), caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), worldwide, including nearly 6 million deaths, since its initial appearance in China in 2019. While the number of diagnosed cases continues to increase, the need for technologies that can accurately and rapidly detect SARS‐CoV‐2 virus infection at early phases continues to grow, and the Federal Drug Administration (FDA) has licensed emergency use authorizations (EUAs) for virtually hundreds of diagnostic tests based on nucleic acid molecules and antigen–antibody serology assays. Among them, the quantitative real‐time reverse transcription PCR (qRT‐PCR) assay is considered the gold standard for early phase virus detection. Unfortunately, qRT‐PCR still suffers from disadvantages such as the complex test process and the occurrence of false negatives; therefore, new nucleic acid detection devices and serological testing technologies are being developed. However, because of the emergence of strongly infectious mutants of the new coronavirus, such as Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529), the need for the specific detection of mutant strains is also increasing. Therefore, this article reviews nucleic acid‐ and antigen–antibody‐based serological assays, and compares the performance of some of the most recent FDA‐approved and literature‐reported assays and associated kits for the specific testing of new coronavirus variants.

Ultrasound-Targeted Microbubble Destruction-Mediated miR-1228 Downregulation Suppresses Tumor Proliferation, Migration, and Invasion of Cervical-Cancer Cells

Objectives: Ultrasound-targeted microbubble destruction (UTMD) is an effective technology for microRNA (miRNA) delivery. miR-1228 plays a crucial role and acts as an oncogenic role in several types of cancers. This study aimed to investigate the functional effect of UTMD-mediated miR-1228 knockdown in cervical-cancer cells. Design: A total of 131 patients who were diagnosed with cervical cancer by histopathological examination were enrolled in this study at the Third Affiliated Hospital of Qiqihar Medical University from February 2018 to January 2021. Participants/Materials, Setting, Methods: miR-1228 expression was tested by reverse-transcription quantitative PCR assay. miR-1228 inhibitors were transfected into cervical-cancer cells using the UTMD method. Then, Cell Counting Kit-8 and transwell assays were carried out to explore the cell proliferation, migration, and invasion potentials, respectively. Results: The results revealed that miR-1228 expression was high in human cervical-cancer tissues and cell lines. Knockdown of miR-1228 suppressed tumor cell proliferation abilities, migration, and invasion capacities. Moreover, UTMD-mediated mIR-1228 inhibitor delivery enhanced the transfection efficiency of miR-1228 inhibitor alone. The UTMD-mediated miR-1228 inhibition enhanced the suppressive role of miR-1228 downregulation on cell proliferation capacity, migration, and invasion abilities in tumor cells, compared to miR-1228 knockdown alone. Limitations: The experiments were carried out only in SiHa and HeLa cells in vitro, and the results were not verified in animals. Conclusions: These results indicated that the delivery of the UTMD-mediated miR-1228 inhibitor might be a potential therapeutic method for the treatment of cervical cancer through suppressing cellular activities.

Irisin Protects Cerebral Neurons from Hypoxia/Reoxygenation via Suppression of Apoptosis and Expression of Pro-Inflammatory Cytokines

Background: Ischemic stroke is a major health issue that causes high incidents of morbidity and mortality worldwide. Irisin is an excise-induced protein that has exhibited pleiotropic properties. Accumulating evidence reveals its critical roles in the regulation of various cellular functions, including nervous system functions. This study aims to disclose the effect of irisin on rat cerebral neurons suffering from hypoxia/reoxygenation (H/R) treatment and to explore the potential underlying molecular mechanisms. Methods: The percentage of rat cerebral neuron cell death was determined by flow cytometry analysis and MTT assay. The expression levels of target genes were measured by western blotting and real-time quantitative reverse transcription PCR assay. Results: Our results demonstrated that irisin treatment substantially reduced H/R-induced apoptosis of rat cerebral neurons. Further investigation revealed that irisin treatment markedly decreased mitogen-activated protein kinase (MAPK) signaling pathway activation and suppressed pro-informatory cytokine expression in cerebral neurons with H/R challenge. Finally, we showed that the neuroprotective effect and anti-inflammatory effect of irisin were comparable with three MAPK signaling inhibitors. Conclusion: Irisin exerts profound neuroprotective and anti-inflammatory effects on H/R-stimulated cerebral neurons by inhibiting the MAPK signaling activation. Therefore, irisin may serve as a potential drug for the treatment of patients with ischemic stroke.

FABP7 inhibits proliferation and invasion abilities of cutaneous squamous cell carcinoma cells via the Notch signaling pathway.

Cutaneous squamous cell carcinoma (CSCC) is one of the most common non-melanoma skin cancers worldwide. Fatty acid-binding protein 7 (FABP7) has been reported to be involved in the occurrence, development, metastasis and prognosis of various tumors. In addition, downregulated FABP7 expression was demonstrated in cutaneous malignant melanoma in a previous study. Therefore, we speculated that FABP7 may be a biomarker for CSCC diagnosis. The aim of the present study was to determine the molecular mechanism underlying the effects of FABP7 in CSCC, which may provide a new diagnostic biomarker or treatment target for CSCC. Reverse transcription-PCR, western blotting and immunohistochemistry assays were performed to detect the expression levels of FABP7 in CSCC tissues and cells. Overexpression of FABP7 was achieved in A431 and colo-16 cell lines by transfection with an overexpression vector (oeFABP7). Cell proliferation, colony formation, migration and invasion were detected by Cell Counting Kit-8, crystal violet, scratch and Transwell assays, respectively. Following FABP7 overexpression, western blotting was used to determine the expression levels of proliferation-, invasion- and Notch pathway-associated proteins, including Snail, N-cadherin, Twist, matrix metalloproteinase (MMP)-2, MMP-7, Notch 1 and Notch 3. In addition a CSCC model in nude mice was constructed. Immunohistochemistry was used to determine the expression levels of FABP7, Ki67, Notch 1 and Notch 3. It was demonstrated that FABP7 expression levels were significantly reduced in human CSCC tissues and cells compared with normal samples. Overexpression of FABP7 inhibited the proliferation, invasion and migration abilities of A431 and colo-16 cells compared with those in the negative control group. In addition, transfection with oeFABP7 reduced the expression levels of proliferation-, invasion- and Notch pathway-associated proteins compared with those in the negative control group. Overexpression of FABP7 also reduced the growth of CSCC tumors in vivo and inhibited the expression of Ki67, Notch 1 and Notch 3. Therefore, the results of the present study suggested that FABP7 may inhibit the proliferation and invasion of CSCC cells via the Notch signaling pathway.

A reverse-transcription droplet digital PCR assay to detect and quantify SARS-CoV-2 RNA in upper respiratory tract specimens.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). Symptoms are variable and range from asymptomatic or mild to severe (i.e., pneumonia) in both healthy and immunocompromised patients. We developed a reverse-transcription droplet digital PCR (RT-ddPCR) assay for quantification of SARS-CoV-2 RNA in clinical nasopharyngeal and oropharyngeal swab specimens and evaluated the assay, including reproducibility, agreement of results, analytical measurement range, linearity, analytical sensitivity, and analytical specificity. This quantitative assay had a LoD of 218 copies/mL of viral transport media, with a linear quantification range from 500 to 5,000,000 copies/mL (R2 of 0.9817 and 0.9853 for N1 and N2 targets, respectively). Qualitative agreement of categorical results was 90.5% (57/63) between the reference and RT-ddPCR assays. Quantitative agreement between the two assays showed correlation, with R2 of 0.9726 and 0.9713 for N1 and N2 targets, respectively. No cross-reactivity with common coronavirus strains was detected. This SARS-CoV-2 quantitative RT-ddPCR assay may be a useful tool for a variety of applications including identification of patients with low viral load and serial monitoring of viral load in respiratory tracts specimens of patients for evaluation of the efficacy of therapy for COVID-19.

MAP9 Exhibits Protumor Activities and Immune Escape toward Bladder Cancer by Mediating TGF-β1 Pathway.

To investigate more potential targets for the treatment of human bladder cancer, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and high-content screening (HCS) analysis were performed, and microtubule-associated protein 9 (MAP9), which had the strongest proliferation inhibition from 809 downregulated genes, has been selected. MAP9 is responsible for bipolar spindle assembly and is involved in the progression of many types of tumors; however, its role in bladder cancer (BC) remains unknown. Expressive levels of MAP9 in BC tissues were determined through immunohistochemistry, and the clinical significance of MAP9 in BC was analyzed. Short hairpin ribonucleic acid- (ShRNA-) MAP9 was used to construct stable MAP9 knockdown BC cell lines. The proliferative abilities of MAP9 were measured through assays in vivo and in vitro, and the migrated and invasive abilities of MAP9 were analyzed via in vitro experiments. Quantitative reverse transcription PCR, western blotting, coimmunoprecipitation (Co-IP), and rescue assays were used to identify downstream targets of MAP9. MAP9 expression increased in the tumor tissues, and its increased level was negatively correlated with prognosis. Further, the loss of MAP9 caused decreased BC cell proliferation via inducing the growth 1/synthesis (G1/S) cell cycle arrest in vitro and slowed tumor growth in vivo. In addition, MAP9 silencing attenuated BC cell migration and invasion. Moreover, we found that the growth 1/synthesis (G1/S) cell cycle-related genes and the epithelial mesenchymal transition (EMT) marker levels decreased after silencing MAP9. Finally, we found that the transforming growth factor beta 1 (TGF-β1) pathway is activated as a mediator for MAP9 to regulate genes related to the G1/S cell cycle and EMT. MAP9 promotes BC progression and immune escape activity through the TGF-β1 pathway and is a potential novel target for therapies of BC.

Duplex One-Step RT-qPCR Assays for Simultaneous Detection of Genomic and Subgenomic RNAs of SARS-CoV-2 Variants.

A hallmark of severe acute respiratory syndrome virus (SARS-CoV-2) replication is the discontinuous transcription of open reading frames (ORFs) encoding structural virus proteins. Real-time reverse transcription PCR (RT-qPCR) assays in previous publications used either single or multiplex assays for SARS-CoV-2 genomic RNA detection and a singleplex approach for subgenomic RNA detection. Although multiplex approaches often target multiple genomic RNA segments, an assay that concurrently detects genomic and subgenomic targets has been lacking. To bridge this gap, we developed two duplex one-step RT-qPCR assays that detect SARS-CoV-2 genomic ORF1a and either subgenomic spike or subgenomic ORF3a RNAs. All primers and probes for our assays were designed to bind to variants of SARS-CoV-2. In this study, our assays successfully detected SARS-CoV-2 Washington strain and delta variant isolates at various time points during the course of live virus infection in vitro. The ability to quantify subgenomic SARS-CoV-2 RNA is important, as it may indicate the presence of active replication, particularly in samples collected longitudinally. Furthermore, specific detection of genomic and subgenomic RNAs simultaneously in a single reaction increases assay efficiency, potentially leading to expedited lucidity about viral replication and pathogenesis of any variant of SARS-CoV-2.

Precise and comprehensive determination of multiple body fluids by applying statistical cutoff values to a multiplex reverse transcription-PCR and capillary electrophoresis procedure for forensic purposes

Body fluid identification from crime scene evidence is an essential procedure in forensic investigations. Among various procedures, multiplex reverse transcription PCR assays have a clear advantage over conventional methods because different types of body fluids can be analyzed simultaneously. For more precise, comprehensive, and objective identification of forensically relevant body fluids, 15 target genes for blood, saliva, semen, vaginal fluid, and nasal secretion were selected; their primers were re-designed and multiplex PCR conditions were optimized to prioritize specificity for those body fluids. Multiple amplicons were separated and determined by the SeqStudio Genetic Analyzer with an all-in-one and easy-to-use cartridge. Then, the cutoff value was set for each marker to eliminate the detection of slight amplification in non-targeted body fluids. As a result, the targeted body fluid specificities of the developed procedure were drastically improved. Although successful determination of the target gene depends on sample condition and marker sensitivity, our procedure was applicable for the precise determination of body fluids in mixed body fluid stains, aged samples, and various mock casework samples. Therefore, it could be a powerful and convenient tool for the precise identification of multiple body fluids in forensic laboratories.

Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of goose astrovirus genotypes 1 and 2

Goose astrovirus (GAstV) is a novel pathogen that was discovered in 2018. It has two genotypes, GAstV-1 and GAstV-2, and both can cause visceral gout of goslings and result in significant economic losses. The present work aimed to develop a duplex TaqMan real-time quantitative reverse transcription PCR (RT-qPCR) assay to distinguish the two genotypes. MegAlign software was used to design two pairs of primers and a pair of matched probes based on the open reading frame 2 (ORF2) sequence with the greatest difference between GAstV-1 and GAstV-2, and primer and probe concentrations and annealing temperatures were optimised. Fluorescence signals were obtained for GAstV-1 and GAstV-2 in the FAM and VIC channels, respectively, but no fluorescent signal was observed for other pathogens. The detection limit for GAstV-1 and GAstV-2 was 33.3 and 33.7 DNA copies/μL, respectively. Intra- and inter-assay variability tests revealed excellent reproducibility. Furthermore, the assay detected GAstV-1 and GAstV-2 in allantoic fluids (100% positive) spiked with viruses, and 70 clinical gout gosling samples were examined, of which 11.4% were positive for GAstV-1, 74.3% were positive for GAstV-2%, and 5.7% were positive for mixed infection. In summary, the developed duplex RT-qPCR assay has high specificity, sensitivity, and reproducibility, and can be used in the clinic for detection of GAstV-1 and GAstV-2.

A novel protein encoded by ZCRB1-induced circHEATR5B suppresses aerobic glycolysis of GBM through phosphorylation of JMJD5

BackgroundRNA-binding proteins (RBPs) and circular RNAs (circRNAs) play important roles in glioblastoma multiforme (GBM). Aerobic glycolysis is a metabolic characteristic of GBM. However, the roles of RBPs and circRNAs in aerobic glycolysis in GBM remain unclear. The aim of this study is to explore the mechanisms by which RBPs and circRNAs regulate aerobic glycolysis in GBM cells.MethodsRNA sequencing and circRNA microarray analysis were performed to identify RBPs and circRNAs for further study. Mass spectrometry validated the encoded protein and its interacting proteins. Quantitative reverse transcription PCR and western blot assays were used to determine the mRNA and protein expression, respectively. Furthermore, immunofluorescence and fluorescence in situ hybridization assays were used to determine the protein and RNA localization, respectively. Glucose and lactate measurement assays, Seahorse XF glycolysis stress assays and cell viability assays were conducted to investigate the effects on glycolysis and proliferation in GBM cells.ResultsWe selected zinc finger CCHC-type and RNA-binding motif 1 (ZCRB1) and circRNA HEAT repeat containing 5B (circHEATR5B) as candidates for this study. These genes were expressed at low levels in GBM tissues and cells. Both ZCRB1 and circHEATR5B overexpression suppressed aerobic glycolysis and proliferation in GBM cells. ZCRB1 overexpression promoted the Alu element-mediated formation of circHEATR5B. In addition, circHEATR5B encoded a novel protein HEATR5B-881aa which interacted directly with Jumonji C-domain-containing 5 (JMJD5) and reduced its stability by phosphorylating S361. JMJD5 knockdown increased pyruvate kinase M2 (PKM2) enzymatic activity and suppressed glycolysis and proliferation in GBM cells. Finally, ZCRB1, circHEATR5B and HEATR5B-881aa overexpression inhibited GBM xenograft growth and prolonged the survival time of nude mice.ConclusionsThis study reveals a novel mechanism of regulating aerobic glycolysis and proliferation in GBM cells through the ZCRB1/circHEATR5B/HEATR5B-881aa/JMJD5/PKM2 pathway, which can provide novel strategies and potential targets for GBM therapy.

Rapid and Accurate Identification of SARS-CoV-2 Variants Using Real Time PCR Assays.

BackgroundGenomic surveillance efforts for SARS-CoV-2 are needed to understand the epidemiology of the COVID-19 pandemic. Viral variants may impact routine diagnostic testing, increase viral transmissibility, cause differences in disease severity, have decreased susceptibility to therapeutics, and/or confer the ability to evade host immunity. While viral whole-genome sequencing (WGS) has played a leading role in surveillance programs, many laboratories lack the expertise and resources for performing WGS. This study describes the performance of multiplexed real-time reverse transcription-PCR (RT-PCR) assays for identification of SARS-CoV-2 variants.MethodsSARS-CoV-2 specimens were tested for spike-gene variants using a combination of allele-specific primer and allele-specific detection technology (PlexPrime® and PlexZyme®). Targeted detection of spike gene mutations by RT-PCR was compared to variant detection in positive specimens by WGS, including the recently emerged SARS-CoV-2 Omicron variant.ResultsA total of 398 SAR-CoV-2 RT-PCR positive and 39 negative specimens previously tested by WGS were re-tested by RT-PCR genotyping. PCR detection of spike gene mutations N501Y, E484K, and S982A correlated 100% with WGS for the 29 lineages represented, including Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). Incorporating the P681R spike gene mutation also allowed screening for the SARS-CoV-2 Delta variant (B.1.617.2 and AY sublineages). Further sampling of 664 specimens that were screened by WGS between June and August 2021 and then re-tested by RT-PCR showed strong agreement for Delta variant positivity: 34.5% for WGS vs 32.9% for RT-PCR in June; 100% vs 97.8% in August. In a blinded panel of 16 Omicron and 16 Delta specimens, results of RT-PCR were 100% concordant with WGS results.ConclusionsThese data demonstrate that multiplexed real-time RT-PCR genotyping has strong agreement with WGS and may provide additional SARS-CoV-2 variant screening capabilities when WGS is unavailable or cost-prohibitive. RT-PCR genotyping assays may also supplement existing sequencing efforts while providing rapid results at or near the time of diagnosis to help guide patient management.

Genome and Phylogenetic Analysis of Cucumber Green Mottle Mosaic Virus Global Isolates and Validation of a Highly Sensitive RT-qPCR Assay.

The last two decades have seen exponential growth in the international movement of seeds for annual food crops, from a gross U.S. import value of $349 million in 1999 to $1.05 billion in 2019. This has led to the proportionate growth of seedborne pathogens dispersed with seed stocks. One such viral pathogen is cucumber green mottle mosaic virus (CGMMV), a tobamovirus that infects cucurbit crops such as melon, watermelon, cucumber, pumpkin, and squash. The first CGMMV introduction to California occurred in 2013, with subsequent annual outbreaks or detections since then. Here, we describe the use of next-generation sequencing to characterize the full genomes of 25 CGMMV isolates collected between 2013 and 2020 in California, either from CGMMV field detections or seed lots identified as CGMMV positive. We sequenced an additional 31 CGMMV isolates collected in Europe, Israel, and southeast Asia that were provided by industry collaborators. We also performed an in silico nucleotide database search in GenBank for full genome CGMMV sequences to include in all in silico analyses. Based on conserved regions within the coat protein gene, we then developed a quantitative reverse-transcription PCR assay for the sensitive and specific detection of CGMMV in seed and plant samples. Finally, based on our sequence and phylogenetic analysis, our data support that CGMMV has been introduced multiple times into California.

Rapid detection of the emerging tick-borne Tamdy virus by TaqMan-based real-time reverse transcription PCR

Tamdy virus (TAMV) is an emerging zoonotic tick-borne arbovirus in the genus Orthonairovirus. Reports of human infections with TAMV have been increasing and development of a rapid detection assay is thus urgently required. In the present study, singleplex and dual-target real-time reverse transcription PCR (qRT-PCR) assays were established for the detection of TAMV. Sensitivity and specificity were evaluated, which demonstrated high sensitivity for both the singleplex and dual-target qRT-PCR assays with no cross-reaction with common bunyaviruses and tick-borne viruses. The TaqMan-based qRT-PCR methodology established in this study can be employed for epidemiological surveillance and pathogenesis studies of TAMV.

Development and Validation of a Pan-Genotypic Real-Time Quantitative Reverse Transcription-PCR Assay To Detect Canine Distemper Virus and Phocine Distemper Virus in Domestic Animals and Wildlife.

Canine distemper virus (CDV) is an animal morbillivirus belonging to the family Paramyxoviridae and has caused major epizootics with high mortality levels in susceptible wildlife species. In recent years, the documented genetic diversity of CDV has expanded, with new genotypes identified in India, the Caspian Sea, and North America. However, no quantitative real-time PCR (RT-qPCR) that has been validated for the detection of all genotypes of CDV is currently available. We have therefore established and characterized a pan-genotypic probe-based RT-qPCR assay based on the detection of a conserved region of the phosphoprotein (P) gene of CDV. This assay has been validated using virus strains representative of six genotypes of CDV in different sample types, including frozen tissue, formalin-fixed paraffin-embedded tissue sections, and virus isolates. The primers and probe target sequences were sufficiently conserved to also enable detection of the phocine distemper virus strains responsible for epizootics in harbor seals in the North Sea in 1988 and 2002. Comparison with two recently published RT-qPCR assays for CDV showed that under equivalent conditions the primers and probe set reported in this study were more sensitive in detecting nucleic acids from an Asia-4 genotype, which displays sequence variation in primer and probe binding sites. In summary, this validated new pan-genotypic RT-qPCR assay will facilitate screening of suspected distemper cases caused by novel genotypes for which full genome sequences are unavailable and have utility in detecting multiple CDV strains in geographical regions where multiple genotypes cocirculate in wildlife.

The potential role of microRNA as bio markers in canine congestive heart failure

ObjectiveIn our focus regarding microRNA, single‐stranded, small non‐coding RNAs, we aim to understand miRNA's roll and functional importance concerning congestive heart failure (CHF) and myxomatous mitral valve degeneration (MMVD). Characterizing expression profiles of circulating microRNAs via genome‐wide sequencing for dogs with CHF secondary to MMVD.Animals8 healthy client‐owned dogs and 8 middle age‐matched client‐owned dogs with CHF secondary to MMVD.ProceduresBlood samples were collected before administering cardiac medications for the management of CHF. Quantitative reverse transcription PCR (qRT‐PCR) assays were used to validate expression profiles of differentially expressed circulating microRNAs identified from NGS analysis of dogs with CHF.Conclusion and Clinical RelevanceAwaiting data indicating the patterns of microRNA expression are distinctive and denote molecular biomarkers with CHF in dogs with MMV.

Fecal extract from obese horses induces an inflammatory response by murine macrophages in vitro.

To compare the inflammatory response of murine macrophages exposed to the enteric microbiome of obese horses versus nonobese horses. Fecal samples from 12 obese horses (body condition score ≥ 7/9) and 12 nonobese horses (body condition score 4 to 5/9) with similar dietary management. Fecal supernatant was prepared from frozen fecal samples. RAW 264.7 macrophage cells were exposed to the fecal extract. Inflammatory cytokine (interleukin-1β, tumor necrosis factor-α, and interleukin-6) gene expression was quantified via real-time quantitative reverse transcription PCR assay, and cytokine concentration was quantified via ELISA. Lipopolysaccharide was evaluated in fecal extract via chromo-limulus amoebocyte lysate assay. Compared with fecal extracts from nonobese horses, fecal extracts from obese horses presented higher concentrations of lipopolysaccharide and induced a heightened expression of the proinflammatory cytokines interleukin-1β, tumor necrosis factor-α, and interleukin-6 from macrophages. The increased levels of inflammatory markers induced in murine macrophages by the microbiome of obese horses in vitro suggested important differences in the enteric microbial composition of these horses, compared with nonobese horses. Overall, this study showed that the microbiome may play a role in mediating an inflammatory response within the gastrointestinal tract of obese horses. Mechanisms of obesity in the horse have not been fully elucidated. Improved understanding of the pathophysiology of disease will guide future research into potential diagnostic and therapeutic interventions for equine obesity.