-
Virus Research Jul 2023Nipah virus (NiV) is a zoonotic pathogen with airborne transmission and high case fatality rates in humans. There is currently no treatment or vaccine against NiV...
Nipah virus (NiV) is a zoonotic pathogen with airborne transmission and high case fatality rates in humans. There is currently no treatment or vaccine against NiV infection approved for humans or animals, therefore early diagnosis is the key to control any potential outbreaks. In this study, we developed an optimized one-pot assay using recombinase polymerase amplification (RPA) coupled to CRISPR/Cas13a for the molecular detection of NiV. The one-pot RPA-CRISPR/Cas13a assay for NiV detection was specific and did not cross-react against other selected (re)-emerging pathogens. The sensitivity of the one-pot RPA-CRISPR/Cas13a assay for NiV detection can detect as little as 10 cp/μL of total synthetic NiV cDNA. The assay was then validated with simulated clinical samples. The results for the one-pot RPA-CRISPR/Cas13a assay could be visualized with either fluorescence or lateral flow strips for convenient clinical or field diagnostics, providing a useful supplement to the gold-standard qRT-PCR assay for detecting NiV detections.
Topics: Humans; Animals; Recombinases; Sensitivity and Specificity; Nipah Virus; Clustered Regularly Interspaced Short Palindromic Repeats; Polymerase Chain Reaction; Nucleic Acid Amplification Techniques; Nucleotidyltransferases
PubMed: 37178792
DOI: 10.1016/j.virusres.2023.199130 -
Frontiers in Public Health 2023Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) may transmit through airborne route particularly when the aerosol particles remain in enclosed spaces with...
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) may transmit through airborne route particularly when the aerosol particles remain in enclosed spaces with inadequate ventilation. There has been no standard recommended method of determining the virus in air due to limitations in pre-analytical and technical aspects. Furthermore, the presence of low virus loads in air samples could result in false negatives. Our study aims to explore the feasibility of detecting SARS-CoV-2 ribonucleic acid (RNA) in air samples using droplet digital polymerase chain reaction (ddPCR). Active and passive air sampling was conducted between December 2021 and February 2022 with the presence of COVID-19 confirmed cases in two hospitals and a quarantine center in Klang Valley, Malaysia. SARS-CoV-2 RNA in air was detected and quantified using ddPCR and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The comparability of two different digital PCR platforms (QX200 and QIAcuity) to RT-PCR were also investigated. Additionally negative staining transmission electron microscopy was performed to visualize virus ultrastructure. Detection rates of SARS-CoV-2 in air samples using ddPCR were higher compared to RT-PCR, which were 15.2% (22/145) and 3.4% (5/145), respectively. The sensitivity and specificity of ddPCR was 100 and 87%, respectively. After excluding 17 negative samples (50%) by both QX200 and QIAcuity, 15% samples (5/34) were found to be positive both ddPCR and dPCR. There were 23.5% (8/34) samples that were detected positive by ddPCR but negative by dPCR. In contrast, there were 11.7% (4/34) samples that were detected positive by dPCR but negative by ddPCR. The SARS-CoV-2 detection method by ddPCR is precise and has a high sensitivity for viral RNA detection. It could provide advances in determining low viral titter in air samples to reduce false negative reports, which could complement detection by RT-PCR.
Topics: Humans; SARS-CoV-2; COVID-19; RNA, Viral; Viral Load; Real-Time Polymerase Chain Reaction; COVID-19 Testing
PubMed: 37965510
DOI: 10.3389/fpubh.2023.1208348 -
Antiviral Research Jul 2023Despite decades of research, human cytomegalovirus (CMV) continues to contribute to significant morbidity and mortality in transplant settings and remains the leading...
Despite decades of research, human cytomegalovirus (CMV) continues to contribute to significant morbidity and mortality in transplant settings and remains the leading cause of viral congenital infections. Clinical diagnosis of CMV infection and/or reactivation under these settings is completed using real time quantitative polymerase chain reaction (RT-qPCR). This assay performs well but is hampered by poor sensitivity and a lack of standardization among testing facilities. A point-of-care rapid diagnostic to determine CMV viremia could address these issues and improve patient care. In this manuscript, we introduce clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a technology to design and validate a rapid diagnostic for CMV. This system was tested using CMV spiked human saliva and urine samples. Sensitivity of the assay was ∼10 infectious units (IU)/mL. Specificity of the assay was robust and failed to detect other herpesviruses. Collectively, we have designed and validated a rapid diagnostic for CMV that overcomes limitations of the current standard diagnostic. This assay has the potential to be used as a point-of-care screening tool in transplant and neonatal settings.
Topics: Infant, Newborn; Humans; Cytomegalovirus; CRISPR-Cas Systems; Rapid Diagnostic Tests; Cytomegalovirus Infections; Real-Time Polymerase Chain Reaction; DNA, Viral
PubMed: 37150408
DOI: 10.1016/j.antiviral.2023.105624 -
Applied and Environmental Microbiology Oct 2023Nucleic acid-based assays, such as polymerase chain reaction (PCR), that amplify and detect organism-specific genome sequences are a standard method for infectious...
Nucleic acid-based assays, such as polymerase chain reaction (PCR), that amplify and detect organism-specific genome sequences are a standard method for infectious disease surveillance. However, challenges arise for virus surveillance because of their genetic diversity. Here, we calculated the variability of nucleotides within the genomes of 10 human viral species and found that endemic viruses exhibit a high percentage of variable nucleotides (e.g., 51.4% for norovirus genogroup II). This genetic diversity led to the variable probability of detection of PCR assays (the proportion of viral sequences that contain the assay's target sequences divided by the total number of viral sequences). We then experimentally confirmed that the probability of the target sequence detection is indicative of the number of mismatches between PCR assays and norovirus genomes. Next, we developed a degenerate PCR assay that detects 97% of known norovirus genogroup II genome sequences and recognized norovirus in eight clinical samples. By contrast, previously developed assays with 31% and 16% probability of detection had 1.1 and 2.5 mismatches on average, respectively, which negatively impacted RNA quantification. In addition, the two PCR assays with a lower probability of detection also resulted in false negatives for wastewater-based epidemiology. Our findings suggest that the probability of detection serves as a simple metric for evaluating nucleic acid-based assays for genetically diverse virus surveillance.IMPORTANCENucleic acid-based assays, such as polymerase chain reaction (PCR), that amplify and detect organism-specific genome sequences are employed widely as a standard method for infectious disease surveillance. However, challenges arise for virus surveillance because of the rapid evolution and genetic variation of viruses. The study analyzed clinical and wastewater samples using multiple PCR assays and found significant performance variation among the PCR assays for genetically diverse norovirus surveillance. This finding suggests that some PCR assays may miss detecting certain virus strains, leading to a compromise in detection sensitivity. To address this issue, we propose a metric called the probability of detection, which can be simply calculated using a code developed in this study, to evaluate nucleic acid-based assays for genetically diverse virus surveillance. This new approach can help improve the sensitivity and accuracy of virus detection, which is crucial for effective infectious disease surveillance and control.
Topics: Humans; Norovirus; Real-Time Polymerase Chain Reaction; RNA, Viral; Nucleotides; Communicable Diseases; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 37791775
DOI: 10.1128/aem.00331-23 -
Scientific Reports Oct 2023Real-time quantitative polymerase chain reaction (RT-qPCR) is the most common method to determine mRNA expression, and Minimum Information for Publication of RT-qPCR...
Real-time quantitative polymerase chain reaction (RT-qPCR) is the most common method to determine mRNA expression, and Minimum Information for Publication of RT-qPCR Experiments (MIQE) proposes that a panel of reference genes for RT-qPCR is conducive to obtaining accurate results. This study aimed to screen and verify the optimal panel of reference genes in colorectal cancer (CRC) and normal colonic cell lines. In the study, eight candidate reference genes (GAPDH, ACTB, 18S, PPIA, B2M, SDHA, GUSB, and YWHAZ) were selected for RT-qPCR to detect their expression in NCM460, HT29, HCT116, SW480, SW620, DLD-1, LOVO and RKO cell lines. The stability of reference genes and the optimal panel were evaluated by geNorm, NormFinder, and BestKeeper software. As results, the expression levels of candidate reference genes differed in the colonic epithelial cell lines, and the number of optimal panel of reference genes is two. B2M and YWHAZ were the two most stable reference genes for NCM460, HCT116, SW620, LOVO, and RKO cell lines, while only one of B2M and YWHAZ was most stable in HT29 and SW480 cells. In DLD-1 cells, the stability of B2M and YWHAZ ranked 3rd and 6th, PPIA and GUSB were the most stable two. Furthermore, the YWHZA + B2M performed smaller intragroup differences than other panel or single reference gene. In conclusion, this study indicates the optimal panel of reference genes is YWHZA + B2M for the NCM460, HCT116, SW620, LOVO, RKO, SW480, and HT29 cell lines, but it is PPIA + GUSB in DLD-1 cell lines.
Topics: Humans; Early Detection of Cancer; Neoplasms; Cell Line; Genes, Essential; Epithelium; Reference Standards; Real-Time Polymerase Chain Reaction; Gene Expression Profiling
PubMed: 37853035
DOI: 10.1038/s41598-023-45174-4 -
Virus Genes Aug 2023The feline leukemia virus (FeLV) belongs to the Retroviridae family and Gammaretrovirus genus, and causes a variety of neoplastic and non-neoplastic diseases in domestic...
The feline leukemia virus (FeLV) belongs to the Retroviridae family and Gammaretrovirus genus, and causes a variety of neoplastic and non-neoplastic diseases in domestic cats (Felis catus), such as thymic and multicentric lymphomas, myelodysplastic syndromes, acute myeloid leukemia, aplastic anemia, and immunodeficiency. The aim of the present study was to carry out the molecular characterization of FeLV-positive samples and determine the circulating viral subtype in the city of São Luís, Maranhão, Brazil, as well as identify its phylogenetic relationship and genetic diversity. The FIV Ac/FeLV Ag Test Kit (Alere™) and the commercial immunoenzymatic assay kit (Alere™) were used to detect the positive samples, which were subsequently confirmed by ELISA (ELISA - SNAP® Combo FeLV/FIV). To confirm the presence of proviral DNA, a polymerase chain reaction (PCR) was performed to amplify the target fragments of 450, 235, and 166 bp of the FeLV gag gene. For the detection of FeLV subtypes, nested PCR was performed for FeLV-A, B, and C, with amplification of 2350-, 1072-, 866-, and 1755-bp fragments for the FeLV env gene. The results obtained by nested PCR showed that the four positive samples amplified the A and B subtypes. The C subtype was not amplified. There was an AB combination but no ABC combination. Phylogenetic analysis revealed similarities (78% bootstrap) between the subtype circulating in Brazil and FeLV-AB and with the subtypes of Eastern Asia (Japan) and Southeast Asia (Malaysia), demonstrating that this subtype possesses high genetic variability and a differentiated genotype.
Topics: Cats; Animals; Leukemia Virus, Feline; Brazil; Phylogeny; Genotype; Polymerase Chain Reaction; Immunodeficiency Virus, Feline; Cat Diseases
PubMed: 37195404
DOI: 10.1007/s11262-023-01997-x -
The Journal of Molecular Diagnostics :... Nov 2023Bacterial commensals of the human genitourinary tract, Mycoplasma hominis and Ureaplasma species (parvum and urealyticum) can be sexually transmitted, and may cause...
Bacterial commensals of the human genitourinary tract, Mycoplasma hominis and Ureaplasma species (parvum and urealyticum) can be sexually transmitted, and may cause nongonococcal urethritis, pelvic inflammatory disease, and infertility. Mycoplasma hominis and Ureaplasma species may also cause severe invasive infections in immunocompromised patients. Current culture-based methods for Mycoplasma/Ureaplasma identification are costly and laborious, with a turnaround time between 1 and 2 weeks. We developed a high-throughput, real-time multiplex PCR assay for the rapid detection of M. hominis and Ureaplasma species in urine, genital swab, body fluid, and tissue. In total, 282 specimens were tested by PCR and compared with historic culture results; a molecular reference method was used to moderate discrepancies. Overall result agreement was 99% for M. hominis (97% positive percentage agreement and 100% negative percentage agreement) and 96% for Ureaplasma species (96% positive percentage agreement and 97% negative percentage agreement). Specimen stability was validated for up to 7 days at room temperature. This multiplex molecular assay was designed for implementation in a high-complexity clinical microbiology laboratory. With this method, >90 samples can be tested in one run, with a turnaround time of 4 to 5 hours from specimen extraction to reporting of results. This PCR test is also more labor effective and cheaper than the conventional culture-based test, thus improving laboratory efficiency and alleviating labor shortages.
Topics: Mycoplasma hominis; Humans; Ureaplasma; Multiplex Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction; Ureaplasma Infections; Mycoplasma Infections; Sensitivity and Specificity; High-Throughput Screening Assays; DNA, Bacterial; Reproducibility of Results
PubMed: 37683891
DOI: 10.1016/j.jmoldx.2023.07.004 -
Einstein (Sao Paulo, Brazil) 2023This study proposes a strategy for large-scale testing among a large number of people for the early diagnosis of COVID-19 to elucidate the epidemiological situation....
This study proposes a strategy for large-scale testing among a large number of people for the early diagnosis of COVID-19 to elucidate the epidemiological situation. Pool testing involves the analysis of pooled samples. This study aimed to discuss a reverse transcription technique followed by quantitative real-time polymerase chain reaction using pool testing to detect SARS-CoV-2 in nasopharyngeal swab samples. The study proposes an innovative diagnostic strategy that contributes to resource optimization, cost reduction, and improved agility of feedback from results. Pool testing is simultaneously performed on multiple samples to efficiently and cost-effectively detect COVID-19. Pool testing can optimize resource utilization and expand diagnostic access, and is a viable alternative for developing countries with limited access to testing. To optimize resources, the pool size was determined by estimating COVID-19 prevalence in the study population.
Topics: Humans; COVID-19; SARS-CoV-2; COVID-19 Testing; Clinical Laboratory Techniques; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 37436266
DOI: 10.31744/einstein_journal/2023AE0115 -
Archives of Oral Biology Oct 2023This study aimed to standardize a quantitative polymerase chain reaction (qPCR)-based test to identify and quantify the uncultivable bacteria associated with...
OBJECTIVE
This study aimed to standardize a quantitative polymerase chain reaction (qPCR)-based test to identify and quantify the uncultivable bacteria associated with periodontitis.
METHODS
The standardization of qPCR, the curves for the quantification of Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis, and Filifactor alocis were developed by cloning the 16 S rRNA target gene fragment, using the GEMTEasy vector. The qPCRs were validated in 55 subgingival biofilm clinical samples, from different stages of periodontitis and from periodontally healthy/gingivitis individuals, which were previously evaluated by next-generation sequencing (NGS). The results obtained by the two methods were compared by the concordance of Cohen's Kappa index, and sensitivity, specificity, receiver operating characteristic (ROC) curve, and predictive values were established.
RESULTS
obtained by the two methods were compared using the concordance of Cohen's Kappa index, and sensitivity, specificity, predictive values, and ROC curves were generated. The qPCR test was standardized with efficiencies between 90% and 100% and R: 0.997-0.999. Concordance between the qPCR and NSG was moderate to F. alocis (agreement 78.2%; kappa 0.56, p < 0.05) and fair to the other microorganisms (agreement 67.27%-72.73; kappa 0.37-0.38, p < 0.05). qPCR exhibited a high sensitivity (82.2-100%) and specificity (100%) for E. brachy, E. saphenum, and F. alocis. Sensitivity was lower to D. oralis. Conversely, qPCR demonstrated higher sensitivity to E. saphenum than NSG (100 vs. 68.1).
CONCLUSIONS
The uncultivable microorganisms associated with periodontitis, D. oralis, E. brachy, E. saphenum, and F. alocis can be detected and quantified with the newly developed and validates qPCR test.
Topics: Humans; Porphyromonas gingivalis; Periodontitis; Gingivitis; Polymerase Chain Reaction
PubMed: 37419062
DOI: 10.1016/j.archoralbio.2023.105758 -
Haematologica Feb 2024Innovations in molecular diagnostics have often evolved through the study of hematologic malignancies. Examples include the pioneering characterization of the... (Review)
Review
Innovations in molecular diagnostics have often evolved through the study of hematologic malignancies. Examples include the pioneering characterization of the Philadelphia chromosome by cytogenetics in the 1970s, the implementation of polymerase chain reaction for high-sensitivity detection and monitoring of mutations and, most recently, targeted next- generation sequencing to drive the prognostic and therapeutic assessment of leukemia. Hematologists and hematopath- ologists have continued to advance in the past decade with new innovations improving the type, amount, and quality of data generated for each molecule of nucleic acid. In this review article, we touch on these new developments and discuss their implications for diagnostics in hematopoietic malignancies. We review advances in sequencing platforms and library preparation chemistry that can lead to faster turnaround times, novel sequencing techniques, the development of mobile laboratories with implications for worldwide benefits, the current status of sample types, improvements to quality and reference materials, bioinformatic pipelines, and the integration of machine learning and artificial intelligence into mol- ecular diagnostic tools for hematologic malignancies.
Topics: Humans; Artificial Intelligence; Mutation; Hematologic Neoplasms; Polymerase Chain Reaction; High-Throughput Nucleotide Sequencing
PubMed: 37584286
DOI: 10.3324/haematol.2022.282442