Construction a novel osteoporosis model in immune-deficient mice with natural ageing.

Osteoporosis (OP) predominantly affects elderly individuals. Stem cells show potential for treating OP. However, animal models with normal immune function can eliminate implanted human cells. This study utilized naturally aging NOD/SCID mice, which exhibit immunodeficiency, to create a human osteoporosis model. This approach helps to minimize the premature immune clearance of transplanted allogeneic or xenogeneic cells in preclinical studies, allowing for a more accurate replication of the clinical pharmacological and pharmacokinetic processes involved in stem cell interventions for osteoporosis. NOD/SCID mice were fed until 12, 32, and 43 weeks of age, respectively, and then euthanized. We harvested lumbar vertebra for Micro-Computed Tomography (Micro-CT) scanning and pathological examination. Additionally, we performed biomechanical testing of lumbar vertebra to assess the severity of osteoporosis. We utilized real-time RT-PCR to assess gene expression changes associated with bone metabolism, aging, inflammation, oxidative stress, and the Tgf-ß1/Smad3 signaling pathway. In addition, the protein expression levels of P16, Tgf-ß1 and Smad3 were detected using Western Blotting (WB). In comparison to 12-week-old mice, the 32-week-old and 43-week-old mice displayed significantly sparser and fractured trabeculae in their lumbar vertebra, lower bone mineral density (BMD), and changes in bone microstructural parameters (∗∗P < 0.01, ∗∗∗P < 0.001). Additionally, compared to 12-week-old mice, the 32-week-old and 43-week-old mice exhibited decreased expression of osteogenic genes (Alp, Opg, Sp7, Col1a1), increased expression of osteoclastic gene (Rankl), the number of TRAP-positive osteoclasts significantly increased in 32-week-old and 43-week-old mice compared to 12-week-old mice. The expression of genes related to aging and inflammatory (P16, Il-1ß, Tnf-α) increases with advancing age (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). The expression of oxidative stress-related genes (Sod1, Sod2, Foxo3, Nrf2), as well as Tgf-ß1 and Smad3 decreased with age (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). As age increases, the levels of P16 protein increase, Tgf-ß1 and Smad3 proteins decrease. Our study successfully replicated osteoporosis models in NOD/SCID mice at both 32 and 43 weeks, with the latter exhibiting more severe osteoporosis. This condition seems to be driven by factors such as aging, inflammation, oxidative stress, and the Tgf-ß1/Smad3 signaling pathway.

Molecular characterization and functional analysis of the ecdysone receptor isoform (EcR) from the oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae).

20-Hydroxyecdysone (20E) plays a vital role in a series of biological processes, via the nuclear receptors, EcR/USP by activating the ecdysone regulatory cascade. To clarify the role of EcR during the development of Grapholita molesta, the complementary DNA of ecdysone receptor isoform B1 (GmEcR-B1) was obtained from the transcriptome of G. molesta and verified by PCR. Alignment analysis revealed that the deduced protein sequence of GmEcR-B1 was highly homologous to EcR proteins identified in other lepidopteran species, especially the EcR-B1 isoform in Spodoptera litura. Quantitative real-time PCR showed that GmEcRs was expressed at all test developmental stages, and the expression level of GmEcRs was relatively higher during the period of the 3rd day of fifth instar larvae to 2nd of pupa than those in other stages. Moreover, the messenger RNA of GmEcRs was much more strongly expressed in the Malpighian tubule and epidermis than those in other tissues, which suggests that this gene may function in a tissue-specific manner during larval development. Silencing of GmEcRs could significantly downregulate the transcriptional level of ecdysone-inducible genes and result in increased mortality during metamorphosis and prolonged prepupal duration. Taken together, the present results indicate that GmEcRs may directly or indirectly affect the development of G. molesta.

Development and Evaluation of an In-House Real-Time RT-PCR Targeting nsp10 Gene for SARS-CoV-2 Detection.

The emergence of SARS-CoV-2 mutations poses significant challenges to diagnostic tests, as these mutations can reduce the sensitivity of commonly used RT-PCR assays. Therefore, there is a need to design diagnostic assays with multiple targets to enhance sensitivity. In this study, we identified a novel diagnostic target, the nsp10 gene, using nanopore sequencing. Firstly, we determined the analytical sensitivity and specificity of our COVID-19-nsp10 assay. The COVID-19-nsp10 assay had a limit of detection of 74 copies/mL (95% confidence interval: 48-299 copies/mL) and did not show cross-reactivity with other respiratory viruses. Next, we determined the diagnostic performance of the COVID-19-nsp10 assay using 261 respiratory specimens, including 147 SARS-CoV-2-positive specimens belonging to the ancestral strain and Alpha, Beta, Gamma, Delta, Mu, Eta, Kappa, Theta and Omicron lineages. Using a LightMix E-gene RT-PCR assay as the reference method, the diagnostic sensitivity and specificity of the COVID-19-nsp10 assay were found to be 100%. The median Cp values for the LightMix E-gene RT-PCR and our COVID-19-nsp10 RT-PCR were 22.48 (range: 12.95-36.60) and 25.94 (range 16.37-36.87), respectively. The Cp values of the COVID-19-nsp10 RT-PCR assay correlated well with those of the LightMix E-gene RT-PCR assay (Spearman's ρ = 0.968; p < 0.0001). In conclusion, nsp10 is a suitable target for a SARS-CoV-2 RT-PCR assay.

Modulation of defense genes and phenolic compounds in wild blueberry in response to Botrytis cinerea under field conditions.

Botrytis blight is an important disease of wild blueberry [(Vaccinium angustifolium (Va) and V. myrtilloides (Vm))] with variable symptoms in the field due to differences in susceptibility among blueberry phenotypes. Representative blueberry plants of varying phenotypes were inoculated with spores of B. cinerea. The relative expression of pathogenesis-related genes (PR3, PR4), flavonoid biosynthesis genes, and estimation of the concentration of ten phenolic compounds between uninoculated and inoculated samples at different time points were analyzed. Representative plants of six phenotypes (brown stem Va, green stem Va, Va f. nigrum, tall, medium, and short stems of Vm) were collected and studied using qRT-PCR. The expression of targeted genes indicated a response of inoculated plants to B. cinerea at either 12, 24, 48 or 96 h post inoculation (hpi). The maximum expression of PR3 occurred at 24 hpi in all the phenotypes except Va f. nigrum and tall stem Vm. Maximum expression of both PR genes occurred at 12 hpi in Va f. nigrum. Chalcone synthase, flavonol synthase and anthocyanin synthase were suppressed at 12 hpi followed by an upregulation at 24 hpi. The expression of flavonoid pathway genes was phenotype-specific with their regulation patterns showing temporal differences among the phenotypes. Phenolic compound accumulation was temporally regulated at different post-inoculation time points. M-coumaric acid and kaempferol-3-glucoside are the compounds that were increased with B. cinerea inoculation. Results from this study suggest that the expression of PR and flavonoid genes, and the accumulation of phenolic compounds associated with B. cinerea infection could be phenotype specific. This study may provide a starting point for understanding and determining the mechanisms governing the wild blueberry-B. cinerea pathosystem.

Detection of H275Y oseltamivir resistance gene mutation among Influenza A(H1N1)pdm09 patients by allelic discrimination real-time RT-PCR.

Influenza viruses can mutate genetically and cause a range of respiratory ailments. The H275Y mutation in the neuraminidase (NA) gene reduces the effectiveness of oseltamivir, a widely used drug for the treatment of Influenza A and B virus infection. The World Health Organization (WHO) recommends single-nucleotide polymorphism assays to detect this mutation. The present study aims to estimate the prevalence of H275Y mutation conferring oseltamivir resistance in Influenza A(H1N1)pdm09 virus among hospitalized patients from June 2014 to December 2021. Following the WHO protocol, allelic discrimination real-time RT-PCR was performed for 752 samples. Out of the 752 samples, 1 tested positive for Y275 gene mutation by allelic discrimination real-time RT-PCR. In samples of years 2020 and 2021, neither the H275 nor Y275 genotype was detected. Sequencing of the NA gene of all negative samples showed a mismatch between the NA sequence and the probes used in the allelic discrimination assay. Also, Y275 mutation was detected in only 1 sample from 2020. The prevalence of oseltamivir resistance was estimated as 0.27% among the Influenza A(H1N1)pdm09 patients during 2014-2021. The study highlights that the WHO-recommended probes for detecting H275Y mutation may not be useful to detect 2020 and 2021 circulating strains of Influenza A(H1N1)pdm09, emphasizing the need for continuous monitoring of mutations in the influenza virus.

Identification of SARS-CoV-2 biomarkers in saliva by transcriptomic and proteomics analysis.

The detection of SARS-CoV-2 biomarkers by real time PCR (rRT-PCR) has shown that the sensitivity of the test is negatively affected by low viral loads and the severity of the disease. This limitation can be overcome by the use of more sensitive approaches such as mass spectrometry (MS), which has not been explored for the detection of SARS-CoV-2 proteins in saliva. Thus, this study aimed at assessing the translational applicability of mass spectrometry-based proteomics approaches to identify viral proteins in saliva from people diagnosed with COVID-19 within fourteen days after the initial diagnosis, and to compare its performance with rRT-PCR. After ethics approval, saliva samples were self-collected by 42 COVID-19 positive and 16 healthy individuals. Samples from people positive for COVID-19 were collected on average on the sixth day (± 4 days) after initial diagnosis. Viable viral particles in saliva were heat-inactivated followed by the extraction of total proteins and viral RNA. Proteins were digested and then subjected to tandem MS analysis (LC-QTOF-MS/MS) using a data-dependent MS/MS acquisition qualitative shotgun proteomics approach. The acquired spectra were queried against a combined SARS-CoV-2 and human database. The qualitative detection of SARS-CoV-2 specific RNA was done by rRT-PCR. SARS-CoV-2 proteins were identified in all COVID-19 samples (100%), while viral RNA was detected in only 24 out of 42 COVID-19 samples (57.1%). Seven out of 18 SARS-CoV-2 proteins were identified in saliva from COVID-19 positive individuals, from which the most frequent were replicase polyproteins 1ab (100%) and 1a (91.3%), and nucleocapsid (45.2%). Neither viral proteins nor RNA were detected in healthy individuals. Our mass spectrometry approach appears to be more sensitive than rRT-PCR for the detection of SARS-CoV-2 biomarkers in saliva collected from COVID-19 positive individuals up to 14 days after the initial diagnostic test. Based on the novel data presented here, our MS technology can be used as an effective diagnostic test of COVID-19 for initial diagnosis or follow-up of symptomatic cases, especially in patients with reduced viral load.

Monomeric myeloperoxidase is a specific biomarker for early-stage ovarian cancer.

Background: Ovarian cancer cells are known to express myeloperoxidase (MPO), an oxidant-producing enzyme with a 150 kDa homodimer, consisting of two identical monomers connected by a disulfide bond. Here, we aim to validate monomeric MPO (mMPO) as a biomarker for the early detection of ovarian cancer.Methods: Human ovarian cancer cells, sera from patients at various stages, sera from non-cancer inflammatory gynecological diseases, and healthy volunteers were used. Monomeric and dimeric MPO were measured by ELISA. Receiver operating curves were used to compare the predictive powers of serum dimeric and monomeric MPO to discriminate between samples.Results: The expression of MPO was unique to ovarian cancer cells. Specifically, mMPO was found to be the only form of MPO in all ovarian cancer cell lines. Intriguingly, mMPO was detected in the sera from all patients with ovarian cancer at various stages, but not from healthy individuals. Serum mMPO discriminated between early-stage ovarian cancer, healthy controls, and benign inflammatory gynecologic disorders. In addition, mMPO discriminated between the early and late stages of the disease.Conclusion: This work highlights mMPO as a potential biomarker for early detection of ovarian cancer, which is critically needed.

Prevalence and genetic diversity in bovine parechovirus infecting Japanese cattle.

The first bovine parechovirus (Bo_ParV) was reported in 2021, and currently, only two nearly complete genome sequences of Bo_ParV are available. In this study, we detected Bo_ParVs in 10 out of 158 bovine fecal samples tested using real-time RT-PCR, and Bo_ParVs were isolated from three of these samples using MA104 cells. Analysis of the P1 region revealed that Bo_ParVs shared high pairwise amino acid sequence similarity (≥ 95.7% identity), suggesting antigenic similarity among Bo_ParVs, whereas nucleotide sequence identity values (≥ 84.8%) indicated more variability. A recombination breakpoint was identified in the 2B region, which may influence the evolution of this virus.

Development and clinical validation of a one-step pentaplex real-time reverse transcription PCR assay for detection of hepatitis virus B, C, E, Treponema pallidum, and a human housekeeping gene.

BACKGROUND: With the safety of blood transfusion being a major public health concern, the development of a rapid, sensitive, specific, and cost-effective multiplex PCR assay for simultaneous detection of hepatitis B virus(HBV), hepatitis C virus (HCV), hepatitis E virus (HEV), and Treponema pallidum(T. pallidum) in blood is crucial. METHODS: Five primer pairs and probes were designed towards conserved regions of target genes and used to establish a one-step pentaplex real-time reverse transcription PCR(qRT-PCR) assay for simultaneous detection of HBV, HCV, HEV, T. pallidum, and RNase P(housekeeping gene), providing sample quality check. The clinical performance of the assay was further determined with 2400 blood samples from blood donors and patients in Zhejiang province, and compared the results with commercial singleplex qPCR and serological assays. RESULTS: The 95% limit of detection(LOD) of HBV, HCV, HEV, and T. pallidum were 7.11 copies/µL, 7.65 copies/µL, 8.45 copies/µL, and 9.06 copies/µL, respectively. Moreover, the assay has good specificity and precision. Compared to the singleplex qPCR assay, the novel assay for detecting HBV, HCV, HEV, and T. pallidum presented 100% clinical sensitivity, specificity, and consistency. Several discrepant results between serological and pentaplex qRT-PCR assays were found. Of 2400 blood samples, there were 2(0.08%) HBsAg positive samples, 3(0.13%) anti-HCV positive samples, 29(1.21%) IgM anti-HEV positive samples and 6(0.25%) anti-T. pallidum positive samples proven negative in nucleic acid detection. 1(0.04%) HBV DNA positive sample and 1(0.04%) HEV RNA positive sample were detected negative by serological testing. CONCLUSIONS: The developed pentaplex qRT-PCR is the first assay on simultaneous, sensitive, specific, and reproducible detection of HBV, HCV, HEV, T. pallidum, and RNase P in a single tube. It could detect pathogens in blood during the window period of infection and is a good tool for effectively screening blood donors and early clinical diagnosis.

A multiplex real-time RT-PCR system to simultaneously diagnose 16 pathogens associated with swine respiratory disease.

AIMS: Swine respiratory disease (SRD) is a major disease complex in pigs that causes severe economic losses. SRD is associated with several intrinsic and extrinsic factors such as host health status, viruses, bacteria, and environmental factors. Particularly, it is known that many pathogens are associated with SRD to date, but most of the test to detect those pathogens can be normally investigated only one pathogen while taking time and labor. Therefore, it is desirable to develop rapidly and efficiently detectable methods those pathogens to minimize the damage caused by SRD. METHODS AND RESULTS: We designed a multiplex real-time RT-PCR (RT-qPCR) system to diagnose simultaneously 16 pathogens, including nine viruses and seven bacteria associated with SRD, on the basis of single qPCR and RT-qPCR assays reported in previous studies. Multiplex RT-qPCR system we designed had the same ability to single RT-qPCR without significant differences in detection sensitivity for all target pathogens at minimum to maximum genomic levels. Moreover, the primers and probes used in this system had highly specificity because the sets had not been detected pathogens other than the target and its taxonomically related pathogens. Furthermore, our data demonstrated that this system would be useful to detect a causative pathogen in the diagnosis using oral fluid from healthy pigs and lung tissue from pigs with respiratory disorders collected in the field. CONCLUSIONS: The rapid detection of infected animals from the herd using our system will contribute to infection control and prompt treatment in the field.

Molecular and serological investigation of Hepatitis E virus in pigs slaughtered in Northwestern Italy.

BACKGROUND: Hepatitis E Virus (HEV) is recently considered an emerging public health concern. HEV genotypes 1 and 2 are widely distributed and pathogenic only for humans. In contrast, HEV, genotypes 3 and 4 are observed in swine, deer, wild boars and rabbits and can also be transmitted to humans. The presence of HEV in the liver, muscle, faeces, blood, and bile was detected by real-time RT-PCR in 156 pigs belonging to twenty different farms, ranging from 1 to 8 months of age. The phylogenetic analysis was performed on the viral strain present in the positive biological matrix, with the lowest Ct. HEV-IgG and HEV-IgM in the sera were analysed by two different ELISA kits. RESULTS: Twenty-one pigs, i.e., 13.46% of them (21/156, 95% CI: 8.53%-19.84%), tested positive for HEV in at least one biological matrix by real-time RT-PCR, while phylogenetic analysis revealed the presence of HEV subtypes 3f and 3c. Pig serums analysed by ELISA showed an overall prevalence of 26.92% (42/156, 95% CI: 20.14%-34.60%) for HEV-IgG, whereas the 28.95% (33/114, 95% CI: 20.84%-38.19%) of them tested negative resulted positive for the HEV-IgM. CONCLUSIONS: The faeces are the biological matrix with the highest probability of detecting HEV. The best concordance value (Kappa Kohen index) and the highest positive correlation (Phi index) were observed for the correlation between bile and liver, even when the number of positive liver samples was lower than the positive bile samples. This finding may suggest that a higher probability of HEV occurs in the bile, when the virus is present in the liver, during the stages of infection. Finally, the presence of HEV in muscle was observed in 11 pigs, usually used for the preparation of some dishes, typical of the Italian tradition, based on raw or undercooked meat. Therefore, their consumption is a possible source of infection for final consumer.

Burden of human metapneumovirus infections among children with acute respiratory tract infections attending a Tertiary Care Hospital, Kathmandu.

BACKGROUND: Acute respiratory infections (ARIs) are one of the most common causes of mortality and morbidity worldwide. Every year millions of children suffer from viral respiratory tract infections (RTIs) ranging from mild to severe illnesses. Human Metapneumovirus (HMPV) is among the most frequent viruses responsible for RTIs. However, HMPV infections and their severity among children have not been explored yet in Nepal. PURPOSE: Therefore, the study was focused on HMPV infections and other potential viral etiologies or co-infections using multiplex PCR among children attending Kanti Children's Hospital and assessed the clinical characteristics of the infections as well as found the co-infections. A hospital-based cross-sectional study was designed and a convenience sampling method was used to enroll children of less than 15 years with flu-like symptoms from both outpatients and inpatients departments over three months of the study period. RESULTS: HMPV infection (13.3%) was the most predominant infection among the different viral infections in children with ARIs in Kanti Children's Hospital. The HMPV was more prevalent in the age group less than three years (21.8%). Cough and fever were the most common clinical features present in all children infected with HMPV followed by rhinorrhea, sore throat, and wheezing. HMPV-positive children were diagnosed with pneumonia (42.9%), bronchiolitis (28.5%), upper respiratory tract infections (14.3%), and asthma (14.3%). The prevalence of HMPV was high in late winter (14.3%) followed by early spring (13.5%). CONCLUSIONS: This study provides the baseline information on HMPV and associated co-infection with other respiratory viruses for the differential diagnosis based on molecular methods and also the comparison of clinical presentations among the different respiratory syndromes.

Influence of pH on Inulin Conversion to 2,3-Butanediol by Bacillus licheniformis 24: A Gene Expression Assay.

2,3-Butanediol (2,3-BD) is an alcohol highly demanded in the chemical, pharmaceutical, and food industries. Its microbial production, safe non-pathogenic producer strains, and suitable substrates have been avidly sought in recent years. The present study investigated 2,3-BD synthesis by the GRAS Bacillus licheniformis 24 using chicory inulin as a cheap and renewable substrate. The process appears to be pH-dependent. At pH 5.25, the synthesis of 2,3-BD was barely detectable due to the lack of inulin hydrolysis. At pH 6.25, 2,3-BD concentration reached 67.5 g/L with rapid hydrolysis of the substrate but was accompanied by exopolysaccharide (EPS) synthesis. Since inulin conversion by bacteria is a complex process and begins with its hydrolysis, the question of the acting enzymes arose. Genome mining revealed that several glycoside hydrolase (GH) enzymes from different CAZy families are involved. Five genes encoding such enzymes in B. licheniformis 24 were amplified and sequenced: sacA, sacB, sacC, levB, and fruA. Real-time RT-PCR experiments showed that the process of inulin hydrolysis is regulated at the level of gene expression, as four genes were significantly overexpressed at pH 6.25. In contrast, the expression of levB remained at the same level at the different pH values at all-time points. It was concluded that the sacC and sacA/fruA genes are crucial for inulin hydrolysis. They encode exoinulinase (EC 3.2.1.80) and sucrases (EC 3.2.1.26), respectively. The striking overexpression of sacB under these conditions led to increased synthesis of EPS; therefore, the simultaneous production of 2,3-BD and EPS cannot be avoided.

Mycobacterium vaccae NCTC 11659, a Soil-Derived Bacterium with Stress Resilience Properties, Modulates the Proinflammatory Effects of LPS in Macrophages.

Inflammatory conditions, including allergic asthma and conditions in which chronic low-grade inflammation is a risk factor, such as stress-related psychiatric disorders, are prevalent and are a significant cause of disability worldwide. Novel approaches for the prevention and treatment of these disorders are needed. One approach is the use of immunoregulatory microorganisms, such as Mycobacterium vaccae NCTC 11659, which have anti-inflammatory, immunoregulatory, and stress-resilience properties. However, little is known about how M. vaccae NCTC 11659 affects specific immune cell targets, including monocytes, which can traffic to peripheral organs and the central nervous system and differentiate into monocyte-derived macrophages that, in turn, can drive inflammation and neuroinflammation. In this study, we investigated the effects of M. vaccae NCTC 11659 and subsequent lipopolysaccharide (LPS) challenge on gene expression in human monocyte-derived macrophages. THP-1 monocytes were differentiated into macrophages, exposed to M. vaccae NCTC 11659 (0, 10, 30, 100, 300 µg/mL), then, 24 h later, challenged with LPS (0, 0.5, 2.5, 250 ng/mL), and assessed for gene expression 24 h following challenge with LPS. Exposure to M. vaccae NCTC 11659 prior to challenge with higher concentrations of LPS (250 ng/mL) polarized human monocyte-derived macrophages with decreased IL12A, IL12B, and IL23A expression relative to IL10 and TGFB1 mRNA expression. These data identify human monocyte-derived macrophages as a direct target of M. vaccae NCTC 11659 and support the development of M. vaccae NCTC 11659 as a potential intervention to prevent stress-induced inflammation and neuroinflammation implicated in the etiology and pathophysiology of inflammatory conditions and stress-related psychiatric disorders.

Molecular detection of SARS-CoV-2 in Argentina: Evaluation of alternative diagnostic tools for the decentralization of the diagnosis.

The rocketing number of COVID-19 cases highlighted the critical role that diagnostic tests play in medical and public health decision-making to contain and mitigate the SARS-CoV-2 pandemic. This study reports the evaluation and implementation of different tests for the molecular detection of SARS-CoV-2 in the central region of Argentina. We evaluated 3 real time RT-PCR kits (GeneFinder COVID-19 Plus RealAmp Kit, DisCoVery SARS-CoV-2 RT-PCR Detection Kit and WGene SARS-CoV-2 RT Detection), 2 nucleic acid extraction methods [MagaBio plus Virus DNA/RNA Purification Kit II (BioFlux), 35-min vs. 9-min], a pre-analytical reagent (FlashPrep®) and 2 isothermal amplification tests (Neokit Plus and ELA CHEMSTRIP®). The order according to the best performance of the 3 real-time RT-PCR kits evaluated was: DisCoVery>GeneFinderTM>WGene. The 2 RNA extraction methods showed similar good results: MagaBio plus Virus RNA Purification Kit II (BioFlux) 9-min was selected due to its faster performance. FlashPrep® reagent showed excellent results to perform direct RNA detection. Isothermal amplification assays showed acceptable sensitivity and specificity values (>80%), except in samples with Ct>30. Our data show optimal real time RT-PCR kits and alternative molecular methods for SARS-CoV-2 diagnostic. These alternative assays proved to be acceptable for their use in adverse contexts, decentralization, and different epidemiological scenarios, for rapid and accurate SARS-CoV-2 detection.

Comparative analysis of novel and common reference genes in adult tissues of the mussel Mytilus galloprovincialis.

BACKGROUND: Real-time quantitative PCR is a widely used method for gene expression analyses in various organisms. Its accuracy mainly relies on the correct selection of reference genes. Any experimental plan involving real-time PCR needs to evaluate the characteristics of the samples to be examined and the relative stability of reference genes. Most studies in mollusks rely on reference genes commonly used in vertebrates. RESULTS: In this study, we focused on the transcriptome of the bivalve mollusk Mytilus galloprovincialis in physiological state to identify suitable reference genes in several adult tissues. Candidate genes with highly stable expression across 51 RNA-seq datasets from multiple tissues were selected through genome-wide bioinformatics analysis. This approach led to the identification of three genes (Rpl14, Rpl32 and Rpl34), whose suitability was evaluated together with 7 other reference genes commonly reported in literature (Act, Cyp-A, Ef1α, Gapdh, 18S, 28S and Rps4). The stability analyses performed with geNorm, NormFinder and Bestkeeper identified specific either single or pairs of genes suitable as references for gene expression analyses in specific tissues and revealed the Act/Cyp-A pair as the most appropriate to analyze gene expression across different tissues. CONCLUSION: Mytilus galloprovincialis is a model system increasingly used in ecotoxicology and molecular studies. Our transcriptome-wide approach represents the first comprehensive investigation aimed at the identification of suitable reference genes for expression studies in this species.

Performance of nasopharyngeal swab and saliva in detecting Delta and Omicron SARS-CoV-2 variants.

A prospective cohort study was conducted during the Delta and Omicron severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) epidemic waves from paired nasopharyngeal swab (NPS or NP swab) and saliva samples taken from 624 participants. The study aimed to assess if any differences among participants from both waves could be observed and if any difference in molecular diagnostic performance could be observed among the two sample types. Samples were transported immediately to the laboratory to ensure the highest possible sample quality without any freezing and thawing steps before processing. Nucleic acids from saliva and NPS were prospectively extracted and SARS-CoV-2 was detected using a real-time reverse-transcription polymerase chain reaction. All observed results were statistically analyzed. Although the results obtained with NP and saliva agreed overall, higher viral loads were observed in NP swabs regardless of the day of specimen collection in both SARS-CoV-2 epidemic waves. No significant difference could be observed between the two epidemic waves characterized by Delta or Omicron SARS-CoV-2. To note, Delta infection resulted in higher viral loads both in NP and saliva and more symptoms, including rhinorrhea, cough, and dyspnea, whereas Omicron wave patients more frequently reported sore throat. An increase in the mean log RNA of SARS-CoV-2 was observed with the number of expressed symptoms in both waves, however, the difference was not significant. Data confirmed that results from saliva were concordant with those from NP swabs, although saliva proved to be a challenging sample with frequent inhibitions that required substantial retesting.

Multiplex detection of eight different viral enteropathogens in clinical samples, combining RT-PCR technology with melting curve analysis.

BACKGROUND: Early and accurate identification of infection viruses among children can benefit the personalized medical treatment and management, and reduce the future occurrence of serious symptoms. Thus, it is critical to develop a high-throughput multiplex real-time RT-PCR method to improve the accuracy and efficiency in routine clinical lab tests. METHODS: We developed a real time RT-PCR combined with melting curve analysis (RRCMC) method for simultaneous detection of rotavirus A, B, C, norovirus GI and GII, adenovirus, astrovirus and sapovirus. RESULTS: Stool samples were collected from 160 children with acute diarrhea and tested by RRCMC assay. A total of 71 patients were tested positive with norovirus, adenovirus or rotavirus. The RRCMC assay has high specificity. There is no internal cross-reaction among the 8 diarrhea viruses and no cross-reaction of other commonly intestinal pathogens and human genome. The limit detection was ranged from 1 × 102 to 1 × 105 nucleic acid copies/ml for each diarrhea virus. CONCLUSION: The RRCMC method is a suitable rapid clinical test for infectious viruses, with the advantages of high-throughput, low cost, high sensitivity and specificity.

Validation of a SARS-CoV-2 RT-PCR assay: a requirement to evaluate viral contamination in human semen.

RESEARCH QUESTION: Is it possible to validate an accurate and reliable method for direct detection of SARS-CoV-2 by reverse transcription polymerase chain reaction (RT-PCR) in human semen fractions? DESIGN: This qualitative improvement study aimed to provide a prospective validation of SARS-CoV-2 detection in male semen. The SARS-CoV-2 genome was detected by multiplex real-time RT-PCR on patient samples that underwent routine semen analyses for infertility at the Center for Reproductive Medicine at the University Hospital of Clermont-Ferrand. Samples comprised surplus semen collected for treatment with assisted reproductive technology. Seminal fluid and spermatozoa fractions were isolated with density gradient centrifugation and cryopreserved. Positive samples were prepared with a standard of inactivated SARS-CoV-2 particles. RESULTS: The analytical method was validated in both seminal fluid and spermatozoa fractions. In both semen fractions, the assay was repeatable, reproducible and showed high sensitivity with a limit of detection of 0.33 SARS-CoV-2 genome copies/µl. The limit of quantification was 1 copy of the SARS-CoV-2 genome/µl. The method was effective regardless of semen quality (normal and altered sperm parameters), number of spermatozoa or the cryoprotectant media used to freeze spermatozoa. CONCLUSION: This validated RT-PCR assay provided accurate and reliable screening of SARS-CoV-2 in seminal fluid and spermatozoa fractions. This method is essential to ensure protection against viral contamination in the cryobanking process.

Considerations for the selection of tests for SARS-CoV-2 molecular diagnostics.

During the course of 2020, the outbreak of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) spread rapidly across the world. Clinical diagnostic testing for SARS-Cov-2 infection has relied on the real-time Reverse Transcriptase Polymerase Chain Reaction and is considered the gold standard assay. Commercial vendors and laboratories quickly mobilised to develop diagnostic tests to detect the novel coronavirus, which was fundamentally important in the pandemic response. These SARS-Cov-2 assays were developed in line with the Food Drug Administration-Emergency Use Authorization guidance. Although new tests are continuously being developed, information about SARS-CoV-2 diagnostic molecular test accuracy has been limited and at times controversial. Therefore, the analytical and clinical performance of SARS-CoV-2 test kits should be carefully considered by the appropriate regulatory authorities and evaluated by independent laboratory validation. This would provide improved end-user confidence in selecting the most reliable and accurate diagnostic test. Moreover, it is unclear whether some of these rapidly developed tests have been subjected to rigorous quality control and assurance required under good manufacturing practice. Variable target gene regions selected for currently available tests, potential mutation in target gene regions, non-standardized pre-analytic phase, a lack of manufacturer independent validation data all create difficulties in selecting tests appropriate for different countries and laboratories. Here we provide information on test criteria which are important in the assessment and selection of SARS-CoV-2 molecular diagnostic tests and outline the potential issues associated with a proportion of the tests on the market.