TaqMan Quantitative Polymerase Chain Reaction Research Articles

One-step triplex TaqMan quantitative reverse transcription polymerase chain reaction for the detection of feline coronavirus, feline panleukopenia virus, and feline leukemia virus

Background and Aim: Feline coronavirus (FCoV), feline panleukopenia virus (FPV), and feline leukemia virus (FeLV) are prevalent throughout China and significantly threaten cat health. These viruses cause similar manifestations and pathological damage. Rapid and accurate diagnosis depends on detection in the laboratory. This study aimed to establish a reliable and rapid method for accurate detection of FCoV, FPV, and FeLV so that a definite diagnosis can be made and effective measures can be taken to prevent and control viral infection. Materials and Methods: We designed three pairs of specific primers and probes for the detection of FCoV 5′ untranslated region, FPV viral protein 2, and FeLV pol genes. Recombinant plasmid constructs were generated for use as standard plasmid constructs. Optimal reaction conditions, including primer and probe concentrations, reaction cycles, and annealing temperatures, were obtained on the basis of optimization tests. One-step triplex real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was successfully established to simultaneously detect FCoV, FPV, and FeLV. The specificity, sensitivity, and repeatability of the assay were analyzed, and its applicability was validated by testing 1175 clinical samples. Results: One-step triplex RT-qPCR had a high degree of specificity only for the detection of FCoV, FPV, and FeLV; it had high sensitivity with limits of detection of 139.904, 143.099, and 152.079 copies/reaction for p-FCoV, p-FPV, and p-FeLV standard plasmid constructs, respectively, and it had reliable repeatability with 0.06%–0.87% intra-assay coefficients of variations. A total of 1175 clinical samples were examined for FCoV, FPV, and FeLV using triplex RT-qPCR, and the FCoV, FPV, and FeLV positivity rates were 18.47%, 19.91%, and 47.57%, respectively. The clinical sensitivity and specificity of one-step triplex RT-qPCR were 93.07% and 97.99%, respectively. Conclusion: We developed a rapid and reliable one-step triplex RT-qPCR method for the detection of FCoV, FPV, and FeLV, which could be used as a diagnostic tool for clinical monitoring and diagnosis. Keywords: detection method, feline coronavirus, feline leukemia virus, feline panleukopenia virus, multiplex reverse transcription-quantitative polymerase chain reaction.

Prevalence and Characteristics of Plasmodium vivax Gametocytes in Duffy-Positive and Duffy-Negative Populations across Ethiopia.

Plasmodium parasites replicate asexually in human hosts. The proportion of infections that carries gametocytes is a proxy for human-to-mosquito transmissibility. It is unclear which proportion of Plasmodium vivax infections in Duffy-negative populations carries gametocytes. We determined the prevalence and characteristics of P. vivax gametocytes in Duffy-positive and -negative populations across broad regions of Ethiopia. Finger-prick blood samples were collected for microscopic and molecular screening of Plasmodium parasites and Duffy status of individuals. Molecular screening of Plasmodium species and Duffy blood group genotyping was done using SYBR green and the Taqman quantitative polymerase chain reaction method. Of the 447 febrile patients who were shown to be P. vivax smear positive, 414 (92.6%) were confirmed by molecular screening as P. vivax and 16 (3.9%) of them were from Duffy-negative individuals. Of these, 5 of 16 (31.3%) Duffy-negative P. vivax-infected samples were detected with gametocytes. Of the 398 Duffy-positive P. vivax-infected samples, 150 (37.7%) were detected with gametocytes, slightly greater than that in Duffy-negative samples. This study highlights the presence of P. vivax gametocytes in Duffy-negative infections, suggestive of human-to-mosquito transmissibility. Although P. vivax infections in Duffy-negative individuals were commonly associated with low parasitemia, some of these infections were shown to have relatively high parasitemia and may represent a prominent erythrocyte invasion capability of P. vivax, and hidden reservoirs that can contribute to transmission. A better understanding of P. vivax transmission biology and gametocyte function particularly in Duffy-negative populations would aid future treatment and management of P. vivax malaria in Africa.

A novel TaqMan qPCR system for the detection of Diaporthe citri, the causative agent of citrus melanose

Citrus melanose, caused by Diaporthe citri, is one of the most economically significant fungal diseases in China. Given that the application of fungicides is the main technique used for the control of citrus melanose, many studies have focused on enhancing the effectiveness and efficiency of the use of fungicides for the control of this economically significant disease. Here, we developed a TaqMan quantitative polymerase chain reaction (qPCR) protocol for monitoring citrus melanose via detection of suspected infected leaves and airborne conidia of D. citri. A primer pair and TaqMan probe targeting D. citri were designed to amplify a region of the fungal β-tubulin gene. The limits of detection and quantification of our TaqMan qPCR method were 159.57 and 683 copies/μL, respectively. The method detected the DNA equivalent to 47 conidia when fungal conidia were cultured in vitro. To evaluate the feasibility of our TaqMan qPCR method, symptomatic and asymptomatic leaf samples, airborne conidia samples were collected from a citrus orchard and tea garden, respectively. D. citri was detected in leaves with citrus melanose symptoms in an orchard; airborne fungal conidia of D. citri were also detected. These findings suggested that this novel TaqMan qPCR method could be used to detect D. citri both in vivo and in vitro. Our methodology could be applied as an alternative method for monitoring the changes in the incidence of D. citri in orchards.

Development of a qPCR Assay for Species-Specific Detection of the Tar Spot Pathogen Phyllachora maydis

Phyllachora maydis is a fungal plant pathogen that causes tar spot of corn ( Zea mays) in North and South America, causing devastating yield losses under favorable conditions. Although the causal agent is relatively easy to diagnose via macroscopic and microscopic observations, other diseases and conditions, such as insect frass, have been mistaken for tar spot of corn. Furthermore, conidia and ascospores in isolation can be difficult to visually distinguish from other fungi, and the development of signs and symptoms of the disease may not be observed until 12 to 20 days after infection. Therefore, we developed a TaqMan quantitative polymerase chain reaction (qPCR) assay for the detection and quantification of this pathogen to be used for diagnostics and airborne spore quantification. The assay was designed for the internal transcribed spacer region of P. maydis. The specificity of the assay was confirmed and tested against various nontarget Phyllachora species, corn pathogens, endophytes, and P. maydis samples from several states in the Midwest and from Mexico. The detection limit of this assay was determined to be 100 fg of genomic P. maydis DNA. To demonstrate the transferability of this technology, the assay was tested in different labs using various qPCR thermal cyclers. This assay can be used in downstream research involving latency period, disease prediction, and diagnostics. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Association of Lactobacillus, Firmicutes, Bifidobacterium, Clostridium, and Enterococcus with colorectal cancer in Iranian patients

BackgroundColorectal cancer (CRC) is one of the primary causes of cancer-associated deaths worldwide, and growing evidence shows that alteration in the gut microbiota may be a contributing factor to the development and progression of the disease. This study investigates the correlation between CRC and specific intestinal microbiota abundance, including Firmicutes, Lactobacillus, Enterococcus, Clostridium, and Bifidobacterium. Material and methodsIn this study, 100 CRC samples and adjacent normal tissues were obtained from Iranian patients. Afterward, we assessed the abundance of the mentioned bacteria in matched tumor and normal tissue samples from 100 CRC patients, by TaqMan quantitative real-time polymerase chain reaction (qPCR). ResultsMost of the patients (55 %) had grade II cancer (moderately differentiated), followed by grade III (poorly Differentiated) in 19 %, and the distribution of the tumor location was 65 % in the colon and 35 % in the rectum. Our research showed a substantial difference in the relative abundance of specific bacteria in tumors and healthy tissues. To this end, four genera of bacteria, including Bifidobacterium, Lactobacillus, Clostridium, and Firmicutes, exhibited statistically significant reductions in tumor tissues compared to adjacent normal tissue (p < 0.05). Conversely, Enterococcus demonstrated a statistically significant increase in tumor tissue samples (p < 0.05). Noteworthy, statistical analysis revealed a significant relationship between Enterococcus and prior cancer (p < 0.05). ConclusionsThese findings provide significant insight into the complex association between the gut microbiota and CRC and may pave the way for future research on novel screening methods, preventive measures, and therapeutic strategies targeting the gut microbiota in CRC patients.

Validation of a rapid HLA-DQA1*05 pharmacogenomics assay to identify at-risk resistance to anti-tumor necrosis factor therapy among patients with inflammatory bowel disease.

The HLA-DQA1*05 variant (rs2097432) is associated with increased risk of immunogenicity to tumor necrosis factor antagonists, with subsequent resistance to therapy in patients with inflammatory bowel disease. Identification of these patients would optimize personalized therapeutic selection. Genomic DNA was extracted from 80 deidentified samples in an unselected patient population with an unknown rs2097432 genotype. Split sample analysis was performed using a reference laboratory. Primer probes for a TaqMan quantitative polymerase chain reaction (qPCR) assay (Thermo Fisher Scientific) were custom designed. Synthesized genomic-block fragments were used as controls. All qPCR reactions were performed using a TaqMan GTXpress Master Mix (Thermo Fisher Scientific) on the Applied Biosystems 7500 system under fast cycling conditions. Of 80 samples, 50% were wild-type reference genotypes, 22.5% were heterozygous, and 27.5% were homozygous variant calls, comparable to population data. Split analysis samples between 2 independent laboratories were 100% concordant. The detection limit tested across genomic-block controls processed in duplicate was reproducible on sample input from 10 ng titrated down to 1.25 ng across 2 independent runs. Further, analytical specificity assessed with previous wild-type reference and homozygous variant DNA spiked into genomic-block controls produced appropriate heterozygous genotypes. Here we present validation of a lab-developed test for a rapid HLA-DQA1*05 (rs2097432) pharmacogenomics assay targeting a hotspot identified by genome-wide association studies. Targeted genotyping employed here will allow for expeditious personalized therapeutic selection.

Norovirus Disease Among Children &amp;lt;5 Years in 3 Sub-Saharan African Countries: Findings From the Vaccine Impact on Diarrhea in Africa (VIDA) Study, 2015–2018

To address a paucity of data from sub-Saharan Africa, we examined the prevalence, severity, and seasonality of norovirus genogroup II (NVII) among children <5 years old in The Gambia, Kenya, and Mali following rotavirus vaccine introduction. Population-based surveillance was conducted to capture medically-attended moderate-to-severe diarrhea (MSD) cases, defined as a child 0-59 months old passing ≥3 loose stools in a 24-hour period with ≥1 of the following: sunken eyes, poor skin turgor, dysentery, intravenous rehydration, or hospitalization within 7 days of diarrhea onset. Diarrhea-free matched controls randomly selected from a censused population were enrolled at home. Stools from cases and controls were tested for enteropathogens, including norovirus and rotavirus, by TaqMan quantitative polymerase chain reaction (PCR) and conventional reverse transcription PCR. We used multiple logistic regression to derive adjusted attributable fractions (AFe) for each pathogen causing MSD, which takes into consideration the prevalence in both cases and controls, for each site and age. A pathogen was considered etiologic if AFe was ≥0.5. In further analyses focusing on the predominant NVII strains, we compared rotavirus and NVII severity using a 20-point modified Vesikari score and examined seasonal fluctuations. From May 2015 to July 2018, we enrolled 4840 MSD cases and 6213 controls. NVI was attributed to only 1 MSD episode. NVII was attributed to 185 (3.8%) of all MSD episodes and was the sole attributable pathogen in 139 (2.9%); peaking (36.0%) at age 6-8 months with majority (61.2%) aged 6-11 months. MSD cases whose episodes were attributed to NVII alone compared with rotavirus alone were younger (median age, 8 vs 12 months, P < .0001) and had less severe illness (median Vesikari severity score, 9 vs 11, P = .0003) but equally likely to be dehydrated. NVII occurred year-round at all study sites. Infants aged 6-11 months bear the greatest burden of norovirus disease, with NVII predominating. An early infant vaccine schedule and rigorous adherence to guidelines recommended for management of dehydrating diarrhea may offer substantial benefit in these African settings.

MP01-09 GENE DOSAGE CHANGES IN CRYPTORCHID MEN WITH NONOBSTRUCTIVE AZOOSPERMIA

You have accessJournal of UrologyCME1 Apr 2023MP01-09 GENE DOSAGE CHANGES IN CRYPTORCHID MEN WITH NONOBSTRUCTIVE AZOOSPERMIA Aaron Brant, Caroline Kang, Anna Mielnik, Marisol O'Neill, Meade Haller, Peter Schlegel, and Dolores Lamb Aaron BrantAaron Brant More articles by this author , Caroline KangCaroline Kang More articles by this author , Anna MielnikAnna Mielnik More articles by this author , Marisol O'NeillMarisol O'Neill More articles by this author , Meade HallerMeade Haller More articles by this author , Peter SchlegelPeter Schlegel More articles by this author , and Dolores LambDolores Lamb More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003212.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Copy number variations (CNVs) are structural chromosomal defects causing microdeletions or microduplications. CNVs in the dosage-sensitive genes ADCY2, ADCY9, CRKL, KCTD13, RBFOX2, and ZEB2 occur more frequently in men with genitourinary anomalies such as cryptorchidism and hypospadias compared to the general male population. To evaluate the association of these CNVs with spermatogenic failure, the prevalence of CNVs in men with history of cryptorchidism who underwent microdissection testicular sperm extraction (mTESE) for non-obstructive azoospermia (NOA) was defined. METHODS: Whole blood DNA samples were collected from 101 men with cryptorchidism and NOA who presented for (mTESE) and two fertile control men. TaqMan quantitative polymerase chain reaction was used to assess copy numbers of ADCY2, ADCY9, CRKL, KCTD13, RBFOX2, and ZEB2 with each reaction performed in triplicate. Relative quantification values were obtained using CopyCaller Software. Fisher’s exact test was used to compare CNVs in cryptorchid men with NOA compared to CNV prevalence in the general male population from the Database of Genomic Variants. RESULTS: Of the 101 patients, 44 (44%) had bilateral cryptorchidism, 57 (56%) had unilateral cryptorchidism, and 51 (50%) had sperm retrieved with mTESE. Six patients (5.9%) had CNVs detected. Two men had microdeletions of CRKL, one had a microdeletion of ZEB2, and one had a microduplication of ADCY2. Two men with bilateral cryptorchidism had multiple CNVs detected: one had microdeletions of ADCY2, ADCY9, and RBFOX2, and one had microdeletions of ADCY9, CRKL, ZEB2 and a microduplication of KCTD13. Four of the six patients with detected CNVs (67%) had sperm retrieved on mTESE. Compared the general male population, men with cryptorchidism and NOA had higher prevalence of CNVs in ADCY2 (p=0.002), ADCY9 (p=0.004), CRKL (p<0.001), and ZEB2 (p<0.001). CONCLUSIONS: Approximately 6% of adult men with a history of cryptorchidism and NOA had gene dosage changes in ADCY2, ADCY9, CRKL, KCTD13, RBFOX2, or ZEB2, with two-thirds of these men having successful sperm retrieval on mTESE. As many of these CNVs are syndromic, genetic counseling may be warranted for men found to carry them. The functional consequences of these gene dosage changes on adult spermatogenesis remain to be fully elucidated. Source of Funding: n/a © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e5 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Aaron Brant More articles by this author Caroline Kang More articles by this author Anna Mielnik More articles by this author Marisol O'Neill More articles by this author Meade Haller More articles by this author Peter Schlegel More articles by this author Dolores Lamb More articles by this author Expand All Advertisement PDF downloadLoading ...

Partial validation of a TaqMan quantitative polymerase chain reaction for the detection of the three genotypes of Infectious spleen and kidney necrosis virus.

Megalocytiviruses (MCVs) are double-stranded DNA viruses known to infect important freshwater and marine fish species in the aquaculture, food, and ornamental fish industries worldwide. Infectious spleen and kidney necrosis virus (ISKNV) is the type species within the genus Megalocytivirus that causes red seabream iridoviral disease (RSIVD) which is a reportable disease to the World Animal Health Organization (WOAH). To better control the transboundary spread of this virus and support WOAH reporting requirements, we developed and partially validated a TaqMan real-time qPCR assay (ISKNV104R) to detect all three genotypes of ISKNV, including the two genotypes that cause RSIVD. Parameters averaged across 48 experiments used a 10-fold dilution series of linearized plasmid DNA (107-101 copies), carrying a fragment of the three-spot gourami iridovirus (TSGIV) hypothetical protein revealed that the assay was linear over 7 orders of magnitude (107-101), a mean efficiency of 99.97 ± 2.92%, a mean correlation coefficient of 1.000 ± 0.001, and a limit of detection (analytical sensitivity) of ≤10 copies of TSGIV DNA. The diagnostic sensitivity and specificity for the ISKNV104R qPCR assay was evaluated and compared to other published assays using a panel of 397 samples from 21 source populations with different prevalence of ISKNV infection (0-100%). The diagnostic sensitivity and specificity for the ISKNV104R qPCR assay was 91.99% (87.28-95.6; 95% CI) and 89.8% (83.53-94.84). The latent class analysis showed that the ISKNV104R qPCR assay had similar diagnostic sensitivities and specificities with overlapping confidence limits compared to a second TaqMan qPCR assay and a SYBR green assay. This newly developed TaqMan assay represents a partially validated qPCR assay for the detection of the three genotypes of the species ISKNV. The ISKNV104R qPCR assay once fully validated, will serve as an improved diagnostic tool that can be used for ISKNV surveillance efforts and diagnosis in subclinical fish to prevent further spread of MCVs throughout the aquaculture and ornamental fish industries.

HSP70 Expression Signature in Renal Cell Carcinoma: A Clinical and Bioinformatic Analysis Approach.

Heat shock proteins (HSPs) are cytoprotective against stressful conditions, as in the case of cancer cell metabolism. Scientists proposed that HSP70 might be implicated in increased cancer cell survival. This study aimed to investigate the HSP70 (HSPA4) gene expression signature in patients with renal cell carcinoma (RCC) in correlation to cancer subtype, stage, grade, and recurrence, combining both clinicopathological and in silico analysis approaches. One hundred and thirty archived formalin-fixed paraffin-embedded samples, including 65 RCC tissue specimens and their paired non-cancerous tissues, were included in the study. Total RNA was extracted from each sample and analyzed using TaqMan quantitative Real-Time Polymerase Chain Reaction. Correlation and validation to the available clinicopathological data and results were executed. Upregulated HSP70 (HSPA4) gene expression was evident in RCC compared to non-cancer tissues in the studied cohort and was validated by in silico analysis. Furthermore, HSP70 expression levels showed significant positive correlations with cancer size, grade, and capsule infiltration, as well as recurrence in RCC patients. The expression levels negatively correlated with the overall survival (r = -0.87, p < 0.001). Kaplan-Meier curves showed lower survival rates in high HSP70 expressor group compared to the low expressors. In conclusion, the HSP70 expression levels are associated with poor RCC prognosis in terms of advanced grade, capsule infiltration, recurrence, and short survival.

Human Parvovirus B19 May Be a Risk Factor in Myasthenia Gravis with Thymoma

Our previous studies have demonstrated that human parvovirus B19 (B19V) is involved in the pathogenesis of thymic hyperplasia-associated myasthenia gravis (MG). However, more cases need to be assessed to further elucidate the relationship between this virus and thymoma-associated MG. The clinicopathological characteristics, presence of B19V DNA, and B19V VP2 capsid protein expression of 708 cases of thymomas were investigated using nested polymerase chain reaction (PCR), TaqMan quantitative (q) PCR, immunohistochemistry, fluorescent multiplex immunohistochemistry, and electron microscopy. Patients with MG or ectopic germinal centers (GCs) were significantly younger than those without MG (P < 0.0001) or GCs (P = 0.0001). Moreover, significantly more GCs were detected in thymomas associated with MG than in those without MG (P < 0.0001). The results of nested PCR and TaqMan qPCR were consistent, and B19V DNA positivity was only associated with presence of GCs (P = 0.011). Immunohistochemically, positive staining was primarily detected in neoplastic thymic epithelial cells (TECs) and ectopic GCs. The positive rate of B19V VP2 was significantly higher in thymoma with MG or GCs than in thymoma without MG (P = 0.004) or GCs (P = 0.006). Electron microscopy showed B19V particles in the nuclei of neoplastic TECs and B cells from GCs. We conclude that the pathogenesis of MG is closely associated with the presence of GCs, and B19V infection is plausibly an essential contributor to formation of ectopic GCs in thymoma. To the best of the authors' knowledge, this is the first study to elucidate the role of B19V in thymoma-associated MG and provide new ideas for exploring the etiopathogenic mechanism of MG.

Alternative splicing in CEP290 mutant cats results in a milder phenotype than LCACEP290 patients.

The rdAc cat has an intronic mutation in the centrosomal 290 kDa (CEP290) gene resulting in a frameshift and a premature stop codon (c.6960 + 9T > G, p.Ile2321AlafsTer3) predicted to truncate the protein by 157 amino acids. CEP290 mutations in human patients cause a range or phenotypes including syndromic conditions and severe childhood loss of vision while the rdAc cat has a milder phenotype. We sought to further characterize the effect of rdAc mutation on CEP290 expression. TaqMan quantitative real-time polymerase chain reaction assays were used to compare wildtype and truncated transcript levels. Relative protein abundance was analyzed by Western blot. Immunohistochemistry (IHC) was performed to detect CEP290 protein. CEP290 mutant cats show low-level (17.4% of wildtype cats) use of the wildtype splice site and usage of the mutant splice site. Western analysis shows retina from cats homozygous for the mutation has CEP290 protein that likely comprises a combination of both wildtype and truncated protein. IHC detects CEP290 in affected and control retina labeling the region of the interconnecting cilium. The comparably milder phenotype of CEP290 mutant cats is likely due to the retained production of some full-length CEP290 protein with possible functional contributions from presence of truncated protein.

Lack of Association between Epidermal Growth Factor or Its Receptor and Reflux Esophagitis, Barrett's Esophagus, and Esophageal Adenocarcinoma: A Case-Control Study.

The epidermal growth factor (EGF) and its receptor (EGFR) gene-gene interactions were shown to increase the susceptibility to esophageal cancer. However, the role of the EGF/EGFR pathway in the development of gastroesophageal reflux disease (GERD) and its complications (reflux esophagitis (RE), Barrett's esophagus (BE), and esophageal adenocarcinoma (EAC)) remains unclear. This association study is aimed at investigating functional EGF and EGFR gene polymorphisms, their mRNA expression in esophageal tissues, and EGF plasma levels in relation to RE, BE, and EAC development in the Central European population. 301 patients with RE/BE/EAC (cases) as well as 98 patients with nonerosive reflux disease (NERD) and 8 healthy individuals (controls) were genotyped for +61 A>G EGF (rs4444903) and +142285 G>A EGFR (rs2227983) polymorphisms using the TaqMan quantitative polymerase chain reaction (qPCR). In random subgroups, the EGF and EGFR mRNA expressions were analyzed by reverse transcription qPCR in esophageal tissue with and without endoscopically visible pathological changes; and the EGF plasma levels were determined by enzyme-linked immunosorbent assay. None of the genotyped SNPs nor EGF-EGFR genotype interactions were associated with RE, BE, or EAC development (p > 0.05). Moreover, mRNA expression of neither EGF nor EGFR differed between samples of the esophageal tissue with and without endoscopically visible pathology (p > 0.05) nor between samples from patients with different diagnoses, i.e., RE, BE, or EAC (p > 0.05). Nevertheless, the lower EGF mRNA expression in carriers of combined genotypes AA +61 EGF (rs4444903) and GG +142285 EGFR (rs2227983; p < 0.05) suggests a possible direct/indirect effect of EGF-EGFR gene interactions on EGF gene expression. In conclusion, EGF and EGFR gene variants and their mRNA/protein expression were not associated with RE, BE or EAC development in the Central European population.

The first detection of Pneumocystis jirovecii in asthmatic patients post-COVID-19 in Jordan.

Pneumocystis jirovecii pneumonia (PCP), caused by fungal species named P. jirovecii, is a frequent opportunistic infection in those with human immunodeficiency virus infection. However, PCP has been documented in immunocompetent patients. This study aims to determine if P. jirovecii detection occurs in asthma patients following coronavirus disease 2019 (COVID-19) in a Jordanian cohort. Another aim was to evaluate a method of TaqMan quantitative polymerase chain reaction (qPCR) assay to detect P. jirovecii, from sputum samples. The nasopharyngeal swabs were used to detect SARS-CoV-2 and sputum samples were tested for P. jirovecii using real-time qPCR assay. Beta-tubulin (BT) and dihydrofolate reductase (DHFR) genes were the directed targets of P. jirovecii. The results showed that the mean qPCR efficiencies of BT and DHFR were 96.37% and 100.13%, respectively. Three out of 31 included patients (9.7%) had a positive P. jirovecii. All of the three patients had used oral corticosteroids (OCS) in the past 2 months due asthma exacerbation and were treated with OCS for COVID-19. This is the first study based in Jordan to demonstrate that P. jirovecii and COVID-19 can coexist and that it is important to maintain a broad differential diagnosis, especially in immunocompromised patients. Chronic lung disease can be a risk factor for the P. jirovecii colonization possibly due to corticosteroid’s immunosuppression.

Molecular analysis of dominant paranasal sinus bacteria in patients with and without chronic rhinosinusitis.

Recent studies have established the possible role of microbiota in developing various diseases. In this regard, attention has shifted to the evaluation of microbiota changes in the paranasal sinuses and its relationship to chronic rhinosinusitis (CRS), especially CRS with nasal polyposis (CRSwNP). This study aimed to examine the bacterial communities of the sphenoidal sinus in Iranian patients with and without CRS. The investigation included 36 subjects, including 18 patients with CRSwNP who underwent Functional Endoscopic Sinus Surgery (FESS) and 18 non-CRS patients who underwent Endoscopic Endonasal Approach (EEA) for pituitary adenoma. The surgeries were performed under general anesthesia, and the sphenoidal sinus was sampled using sterile rayon-tipped swabs coated with a sheet. TaqMan quantitative real-time polymerase chain reaction (qPCR) method (the 16S rDNA gene from bacteria) was used for detection of bacterial communities in different samples. Staphylococcus haemolyticus and Pseudomonas aeruginosa were significantly more prevalent in CRS patients than non-CRS patients (P value ≤ 0.05). However, no significant difference in the frequency of Corynebacterium spp. and Staphylococcus aureus was observed between the two groups, and no Streptococcus pneumoniae or Haemophilus influenza species were isolated from any of the samples. The current study's findings indicated a significant difference in the frequency of certain bacterial species in patients with CRS vs. non-CRS patients. By establishing a link between microbial burden and CRS, it is possible to develop effective treatments or even prevent disorders in this body area.

The role of ANP32B as a biomarker and therapeutic target in head and neck cancer

<h3>Introduction</h3> Acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) is involved in gene expression, apoptosis, cell proliferation, and cell cycle progression. The role of this protein in head and neck squamous cell carcinoma (HNSCC) pathogenesis is not yet known. <h3>Materials and methods</h3> Biopsy specimens were analyzed from patients with HNSCC, oral cavity dysplasia, and benign tumors using miRNA TaqMan assays and quantitative polymerase chain reaction (n=45). Additionally, a luciferace reporter assay, loss of function and gain of function studies, and The Cancer Genome Atlas (TCGA) were used to perform analyses of gene expression data in relation with clinical data and survival outcomes. <h3>Results</h3> micro-RNA analysis of patients with HNSCC and controls showed an increase in miRNA1-2 expression in patients with oral cancer (P<0.05). A luciferase reporter assay showed the role of miRNA 1-2 in the post translational regula- tion of ANP32B expression. Loss of function and gain of function studies showed a mechanistic role of the miRNA 1-2-ANP32B axis in carcinogenesis and AKT phosphorylation in HNSCC cell lines. Analysis of the Cancer Genome Atlas (TCGA) data showed a significant increase of ANP32B expression in head and neck cancer patients (P<0.05). Levels of ANP32B expression were significantly higher in stage 2-4 compared to stage 1 of HNSCC. Males with head and neck cancer showed a significantly higher expression of ANP32B compared to females (P<0.05). No differential expression of ANP32B between different races was found. ANP32B expression was significantly associated with a higher grade of tumor (P-trend<0.05). Higher expression of ANP32B was found in N3 and N2 metastasis stages versus N1. High expression of ANP32B in females was associated with the poorest prognosis (P<0.05). <h3>Conclusions</h3> Altogether, our findings suggest that ANP32B acts as an oncogene in HNSCC and can be used as a potential thera- peutic target for HNSCC treatment.

Development of a quantitative polymerase chain reaction assay for detection of the aetiological agents of piscine lactococcosis.

Piscine lactococcosis is an emergent bacterial disease that is associated with high economic losses in many farmed and wild aquatic species worldwide. Early and accurate detection of the causative agent of piscine lactococcosis is essential for management of the disease in fish farms. In this study, a TaqMan quantitative polymerase chain reaction (qPCR) targeting the 16S-23S rRNA internal transcribed spacer region was developed and validated. Validation of the qPCR was performed with DNA of previously typed L. petauri and L. garvieae recovered from different aquatic hosts from distinct geographical locations, closely related bacterial species and common pathogens in trout aquaculture. Further diagnostic sensitivity and specificity was investigated by screening of fish, water and faecal samples. The developed qPCR assay showed high specificity, sensitivity and accuracy in detection of L. petauri and L. garvieae with lack of signals from non-target pathogens, and in screening of rainbow trout (Oncorhynchus mykiss) posterior kidney and environmental samples. The detection limit of the qPCR was four amplicon copies. Moreover, the sensitivity of the qPCR assay was not affected by presence of non-target DNA from either fish or environmental samples. The robustness, specificity and sensitivity of the developed qPCR will facilitate fast and accurate diagnosis of piscine lactococcosis to establish appropriate control measures in fish farms and aquaria.

Development and Application of a Triplex TaqMan Quantitative Real-Time PCR Assay for Simultaneous Detection of Feline Calicivirus, Feline Parvovirus, and Feline Herpesvirus 1.

As companion animals, felines play an important role in human's family life, and their healthcare has attracted great attention. Viruses such as feline calicivirus (FCV), feline herpesvirus 1 (FHV-1), and feline parvovirus virus (FPV) are the most common pathogens that cause severe infectious disease in baby cats. Thus, preclinical detection and intervention of these three viruses is an effective means to prevent diseases and minimize their danger condition. In this study, a triplex TaqMan quantitative real-time polymerase chain reaction (qRT-PCR) assay was developed to detect these three viruses simultaneously. The detection limit of FPV, FCV, and FHV-1 was 5 × 101 copies/assay, which exhibited higher sensitivity (about 10- to100-fold) than conventional PCR. The coefficients of variation (CVs) of the intra-assay variability were lower than 1.86%, and that of inter-assay variability were lower than 3.19%, indicating the excellent repeatability and reproducibility of the triplex assay. Additionally, the assay showed good specificity. Finally, samples from 48 cats were analyzed using the established assay and commercial kits. As a result, the total positive rates for these viruses were 70.83 or 62.5%, respectively, which demonstrated that the developed qRT-PCR assay was more accurate than the commercial kits and could be used in clinical diagnosis.

Circulating miR-21-5p, miR-146a-5p, miR-320a-3p in patients with atrial fibrillation in combination with hypertension and coronary artery disease

Aim. To study the plasma profiles of circulating extracellular microribonucleic acids (miRNAs), potentially including in pathogenesis of cardiovascular diseases, in patients with atrial fibrillation (AF) in combination with hypertension (HTN) or coronary artery disease (CAD).Material and methods. The study included patients with AF in combi nation with HTN (n=21) or CAD (n=10), as well as following control groups: patients with uncomplicated HTN without AF (n=28), patients with stable CAD without AF (n=10) and healthy individuals (n=30). MiRNA samples were isolated from blood plasma of the study participants. MiRNAs were detected by TaqMan quantitative polymerase chain reaction assay. The relative plasma levels of five candidate miRNAs were estimated relative to the reference miR-16-5p.Results. Among the analyzed circulating plasma miRNAs, a higher level of miR-320a-3p was associated with AF, while increased levels of miR 146a-5p and miR-21-5p are potentially associated with presence of both AF and CAD.Conclusion. Differences were found in the plasma miRNA profiles (miR-21-5p, miR-320a-3p, miR-146a-5p) between patients with AF, regardless of concomitant disease (CAD or HTN), and healthy individuals in the control group.

Development and Clinical Validation of a Potential Penside Colorimetric Loop-Mediated Isothermal Amplification Assay of Porcine Circovirus Type 3.

Porcine circovirus type 3 (PCV3), a novel circovirus, imposes great burdens on the global pig industry. The penside tests for detecting PCV3 are critical for assessing the epidemiological status and working out disease prevention and control programs due to the unavailability of a commercial vaccine. A one-step molecular assay based on visual loop-mediated isothermal amplification (vLAMP) was developed for simple and rapid detection of PCV3. We compared its sensitivity and specificity with TaqMan quantitative real-time polymerase chain reaction (qPCR) and applied the developed assay in the epidemiological study of (n = 407) pooled swine sera collected from almost the entire mainland China during the years 2017–2018. We also explored the feasibility of the vLAMP assay for detecting raw samples without a prior DNA isolation step to expand its application capability. Results showed that the vLAMP assay could reliably detect the PCV3 cap gene with a detection limit of 10 DNA copies equal to that of the Taqman qPCR assay. In the epidemiological study, the PCV3 positive detection rate for 407 swine pooled sera detected by the vLAMP assay was 37.35% (152/407), whereas it was 39.01% (159/407) for Taqman qPCR. For the detection method without genome extraction, the results kept satisfactory specificity (100%) but displayed lower sensitivity (100% for CT < 32), indicating the direct detection is not sensitive enough to discriminate the samples with low viral loads. The one-step vLAMP is a convenient, rapid, and cost-effective diagnostic for penside detection and will enable the epidemiological surveillance of PCV3, which has widely spread in mainland China.