Specific Reverse Transcription Research Articles

Identification and Molecular Characterization of Shamonda Virus in an Aborted Goat Fetus in South Africa.

Viruses in the Orthobunyavirus genus, Peribunyaviridae family, are associated with encephalitis, birth defects and fatalities in animals, and some are zoonotic. Molecular diagnostic investigations of animals with neurological signs previously identified Shuni virus (SHUV) as the most significant orthobunyavirus in South Africa (SA). To determine if other orthobunyaviruses occur in SA, we screened clinical specimens from animals with neurological signs, abortions, and acute deaths from across SA in 2021 using a small (S) segment Simbu serogroup specific TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR). Positive cases were subjected to Sanger sequencing and phylogenetic analysis to identify specific viruses involved, followed by next-generation sequencing (NGS) and additional PCR assays targeting the medium (M) segment and the large (L) segment. In total, 3/172 (1.7%) animals were PCR positive for Simbu serogroup viruses, including two horses with neurological signs and one aborted goat fetus in 2021. Phylogenetic analyses confirmed that the two horses were infected with SHUV strains with nucleotide pairwise (p-) distances of 98.1% and 97.6% to previously identified strains, while the aborted goat fetus was infected with a virus closely related to Shamonda virus (SHAV) with nucleotide p-distances between 94.7% and 91.8%. Virus isolation was unsuccessful, likely due to low levels of infectious particles. However, phylogenetic analyses of a larger fragment of the S segment obtained through NGS and partial sequences of the M and L segments obtained through RT-PCR and Sanger sequencing confirmed that the virus is likely SHAV with nucleotide p-distances between 96.6% and 97.8%. This is the first detection of SHAV in an aborted animal in SA and suggests that SHAV should be considered in differential diagnosis for abortion in animals in Southern Africa.

Absolute quantification of a plasma tRNA-derived fragment for the diagnosis and prognosis of gastric cancer.

The transition from a healthy gastric mucosa to gastric cancer is a multi-step process. Early screening can significantly improve the survival rate of gastric cancer patients. A reliable liquid biopsy for gastric cancer prediction is urgently needed and since tRNA-derived fragments (tRFs) are abundant in various body fluids, tRFs are possible new biomarkers for gastric cancer. A total of 438 plasma samples from patients with different gastric mucosal lesions as well as healthy individuals were collected. A specific reverse transcription primer, a forward primer, a reverse primer, and a TaqMan probe were designed. A standard curve was constructed and an absolute quantitation method was devised for detection of tRF-33-P4R8YP9LON4VDP in plasma samples of individuals with differing gastric mucosa lesions. Receiver operating characteristic curves were constructed to evaluate the diagnostic values of tRF-33-P4R8YP9LON4VDP for individual with differing gastric mucosa. A Kaplan-Meier curve was established to calculate the prognostic value of tRF-33-P4R8YP9LON4VDP for advanced gastric cancer patients. Finally, a multivariate Cox regression analysis was performed to assess the independent prognostic value of tRF-33-P4R8YP9LON4VDP for advanced gastric cancer patients. A detection method for plasma tRF-33-P4R8YP9LON4VDP was successfully established. Levels of plasma tRF-33-P4R8YP9LON4VDP were shown to reflect a gradient change from healthy individuals to gastritis patients to early and advanced gastric cancer patients. Significant differences were found among individuals with differing gastric mucosa, with reduced levels of tRF-33-P4R8YP9LON4VDP significantly related to a poor prognosis. tRF-33-P4R8YP9LON4VDP was found to be an independent predictor of an unfavorable survival outcome. In this study, we developed a quantitative detection method for plasma tRF-33-P4R8YP9LON4VDP that exhibited hypersensitivity, convenience, and specificity. Detection of tRF-33-P4R8YP9LON4VDP was found to be a valuable means by which to monitor different gastric mucosa and to predict patient prognosis.

Rapid, Sensitive and Simultaneous Detection of Two Wheat RNA Viruses Using Reverse Transcription Recombinase Polymerase Amplification (RT-RPA).

In China, wheat yellow mosaic disease is mostly caused by wheat yellow mosaic virus (WYMV) and Chinese wheat mosaic virus (CWMV). If wheat is co-infected with these two viruses, it can cause severe yellow mosaic symptoms and yield losses. Early detection of viruses is crucial for preventing disease in the field. In this study, we optimized a sensitive, specific reverse transcription recombinase polymerase amplification (RT-RPA) detection method for two viruses, WYMV and CWMV. Two sets of primers were designed based on the capsid protein (CP)-encoding genes of the two viruses, and the reaction conditions were determined. The RT-RPA method, which amplified the target amplicon by a handheld reaction mixture for 20 min, was more sensitive than PCR-CP in the detection of WYMV. Finally, the RT-RPA method was performed on 110 randomly selected field samples, demonstrating its applicability to samples from different regions and specificity for co-infected samples. This study not only describes an improved method for detecting WYMV and CWMV using RT-RPA but also demonstrates the potential of this method, which could be applied under field conditions.

Quantitative Evaluation of Very Low Levels of HIV-1 Reverse Transcriptase by a Novel Highly Sensitive RT-qPCR Assay.

Based on previous experience in our laboratory, we developed a real-time reverse transcriptase (RT) quantitative PCR (RT-qPCR) assay for the assessment of very low levels of HIV-1 RT activity. The RNA, acting as a template for reverse transcription into cDNA by HIV-1 RT, consisted of a synthetic RNA ad hoc generated by in vitro transcription and included a coding sequence for HSV-1 gD (gD-RNA-synt). Different conditions of variables involved in the RT-qPCR reaction, notably different amounts of gD-RNA-synt, different mixes of the reaction buffer, and different dNTP concentrations, were tested to optimize the assay. The results indicated that the gD-RNA-synt-based RT assay, in its optimized formulation, could detect a specific cDNA reverse transcription even in the presence of 1 × 10−9 U of HIV RT. This achievement greatly improved the sensitivity of the assay over previous versions. In summary, this constructed RT-qPCR assay may be considered a promising tool for providing accurate information on very low HIV-1 RT activity.

XNA probe and CRISPR/Cas12a-powered flexible fluorescent and electrochemical dual-mode biosensor for sensitive detection of m6A site-specific RNA modification

N6-methyladenosine (m6A) in RNAs is closely related to various biological progresses, but the specific regulatory mechanisms are still unclear. The existing m6A single-base resolution analysis techniques have problems of specificity and sensitivity to be improved, which can hardly meet the urgent needs of basic research and clinical applications. This work proposes a new strategy based on xeno nucleic acid (XNA) probe and CRISPR/Cas12a signal amplification for the sensitive detection of site-specific m6A modifications. According to the difference in the thermodynamic stability of hybridization between XNA probe with m6A-RNA and A-RNA, XNA was designed as a block probe to mediate m6A-RNA specific reverse transcription polymerase chain reaction (MsRT-PCR). Therefore, m6A can be specifically distinguished by converting difficult-to-test m6A modifications into easily detectable dsDNA fragments. Integration of CRISPR/Cas12a technology, skilfully designed sequences of crRNAs targeting m6A site-specific amplification dsDNA. The specificity was significantly improved through dual specific recognition of XNA probe and crRNA. Furthermore, the sensitivity of the assay was also greatly increased by the combined signal amplification of PCR and CRISPR/Cas12a. Additionally, we extend the application of CRISPR/Cas12a to flexible fluorescent and electrochemical biosensing system, which can accurately detect m6A modifications with different ranges of methylation fractions. The analysis results of m6A sites in MALAT1, ACTB and TPT1 further demonstrated the feasibility of the constructed biosensor for the accurate detection of hypomethylated samples in cells. The implementation of this work will provide strong technical support to promote the in-depth research on m6A in disease regulation mechanisms and in vitro molecular diagnosis.

Transcriptome-wide profiling of N 6-methyladenosine via a selective chemical labeling method.

Studies of chemical modifications on RNA have ushered in the field of epitranscriptomics. N 6-Methyladenosine (m6A) is the most typical RNA modification and is indispensable for basic biological processes. This study presents a chemical pulldown method (m6A-ORL-Seq) for transcriptome-wide profiling of m6A. Moreover, chemical labeling results in a specific reverse transcription (RT) truncation signature. This study has identified four thousand high-confidence m6A sites at single-base resolution in the human transcriptome. Unlike previously reported methods based on m6A-antibody or m6A-sensitive enzymes, the antibody/enzyme-free chemical method provides a new perspective for single-base m6A detection at the transcriptome level.

The Effects of the Uncontrolled Use of Biomodulina T on the Severity of Severe Acute Respiratory Syndrome-Coronavirus 2 Infection in Older Cuban Adults: An Open Label Evaluation

BackgroundThe ongoing coronavirus disease 2019 (COVID-19) pandemic is a worldwide public health threat. Millions of people are at risk and older adults are more susceptible to developing the most serious manifestations of the disease, in part because of the effects of ageing on the immune system. Biomodulina T is an injectable immune modulator that has been licensed for use in Cuba for many years.ObjectiveAn open-label, uncontrolled trial was conducted to investigate whether or not it might be useful to prevent or modulate the serious effects of severe acute respiratory syndrome coronavirus 2 infections in older Cuban adults before the availability of vaccinations.MethodsFrom April 12 to August 31, 2021, 1239 adults aged 60 years and older, unvaccinated against COVID-19 were recruited from the José Luis Dubrocq polyclinics, to receive Biomodulina T, 1 intramuscular 3 mL dose weekly for 6 weeks. Each person was visited at home weekly to be administered Biomodulina T. Once daily patients were seen by a medical student to collect information on any possible adverse events related to the medication as well as any symptoms of COVID-19. The possible usefulness of the intervention and its potential adverse events were assessed based on the number of older adults who became infected with COVID-19, and the severity of any symptoms reported or noted both during the 6-week treatment period and during an additional 6-week posttreatment observation period.ResultsSixteen patients were diagnosed with symptomatic COVID-19 during the intervention using a specific reverse transcription polymerase chain reaction test. One patient died because of COVID-19. The most common preexisting diagnoses in treated patients included high blood pressure in 64.8%, diabetes mellitus in 19.85%, and ischemic cardiopathy in 13.88%. Biomodulina T was well tolerated. Only infrequent, mild, transient, and self-limited adverse events were identified. Both the incidence of COVID-19 infections and the overall mortality rate were lower in the treated patients than what was observed in the untreated general population of this Cuban province during the same time period.ConclusionsAlthough further, confirmatory, double-blind, controlled clinical trials are needed, Biomodulina T injections were well tolerated, and the results of this open, uncontrolled study suggest that it may have been useful to decrease the incidence and severity of symptomatic COVID-19 infection in these older Cuban adults. (Curr Ther Res Clin Exp. 2022; 82:XXX–XXX) © 2022 Elsevier HS Journals, Inc

A Simple Multiplex Reverse Transcription-PCR Method for the Diagnosis of L-A and M Totiviruses in Saccharomyces cerevisiae.

Killer yeasts and their toxins have many potential applications in environmental, medical, and industrial biotechnology. The killer phenotype in Saccharomyces cerevisiae relies on the cytoplasmic persistence of two dsRNA viruses, L-A and M. M encodes the toxin, and L-A provides proteins for expression, replication, and capsids for both viruses. Yeast screening and characterization of this trait are usually performed phenotypically based on their toxin production and immunity. In this study, we describe a simple and specific reverse transcription (RT) multiplex PCR assay for direct diagnosis of the dsRNA totivirus genomes associated with the killer trait in the S. cerevisiae yeast. This method obviates RNA purification steps and primer addition to the RT reaction. Using a mixture of specific primers at the PCR step, this multiplex RT-PCR protocol provided an accurate diagnosis of both L-A and M totivirus in all its known variants, L-A-1/M1, L-A-2/M2, L-A-28/M28, and L-A-lus/Mlus, found in infected killer yeasts. Using this method, the expected L-A-2/M2 totivirus associations in natural wine yeasts cells were identified but, importantly, asymptomatic L-A-2/M2 infected cells were found in addition to unexpected L-A-lus/M2 totiviral associations. IMPORTANCE The killer phenomenon in S. cerevisiae yeast cells provides the opportunity to study host-virus interactions in a eukaryotic model. Therefore, the development of simple methods for their detection significantly facilitates their study. The simplified multiplex RT-PCR protocol described here provides a useful and accurate tool for the genotypic characterization of yeast totiviruses in killer yeast cells. The killer trait depended on two dsRNA totiviruses, L-A and M. Each M dsRNA depends on a specific helper L-A virus. Thus, direct genotyping by the described method also provided valuable insights into L-A/M viral associations and their coadaptational events in nature.

Baiting out a full length sequence from unmapped RNA-seq data

BackgroundAs a powerful tool, RNA-Seq has been widely used in various studies. Usually, unmapped RNA-seq reads have been considered as useless and been trashed or ignored.ResultsWe develop a strategy to mining the full length sequence by unmapped reads combining with specific reverse transcription primers design and high throughput sequencing. In this study, we salvage 36 unmapped reads from standard RNA-Seq data and randomly select one 149 bp read as a model. Specific reverse transcription primers are designed to amplify its both ends, followed by next generation sequencing. Then we design a statistical model based on power law distribution to estimate its integrality and significance. Further, we validate it by Sanger sequencing. The result shows that the full length is 1556 bp, with insertion mutations in microsatellite structure.ConclusionWe believe this method would be a useful strategy to extract the sequences information from the unmapped RNA-seq data. Further, it is an alternative way to get the full length sequence of unknown cDNA.

Toscana Virus: Ten Years of Diagnostics in Portugal.

Toscana virus (TOSV) is an emerging sandfly-borne virus within the Phlebovirus genus. Although most infections caused by this virus present as asymptomatic or with minimal symptomatology, TOSV may emerge as a febrile disease or sporadic cases of neurological disease such as meningitis or meningoencephalitis. This pathogen is distributed throughout the Mediterranean basin, along with the spatial distribution of its recognized sandfly vector, Phlebotomus perniciosus. Portugal, after Italy, was the second country considered endemic for this virus, with the first case of acquired infection published in 1985. Although little is known about the circulation of this virus in Portugal, the laboratory diagnosis of TOSV is available at the Centre for Vectors and Infectious Diseases Research of the National Institute of Health Dr. Ricardo Jorge (CEVDI/INSA), since 2007. The aim of this study is to report the results of the diagnosis of TOSV at the CEVDI/INSA, between 2009 and 2018. The diagnosis of TOSV in the CEVDI/INSA is included in the arboviruses and vector-borne neurotropic viruses panels or can be performed, when specified, for TOSV only. Direct detection is made in cerebrospinal fluid samples and is available for TOSV by specific real-time reverse transcription polymerase chain reaction followed by conventional real-time reverse transcription polymerase chain reaction for sequencing purposes, if positive. For indirect diagnosis, performed in serum samples, an in-house immunofluorescence assay for the detection of IgM and IgG antibodies against TOSV is used. A commercial immunofluorescence assay consisting in a mosaic of four phleboviruses is also available, in which, in addition to TOSV, antibody detection for sandfly fever Naples virus, sandfly fever Sicilian virus and sandfly fever Cyprus virus can be done. All diagnostic tests requested by clinicians to the CEVDI/INSA for arboviruses, neurotropic viruses and/or TOSV between January 2009 and December 2018, were included in this study. During the study period, the CEVDI/INSA received samples from 608 patients with diagnostic requests for TOSV. Five acute TOSV infections and one acute sandfly fever Sicilian virus infection were confirmed in serum samples. Three other patients had serological evidence of previous contact with the virus. Two of the six patients with acute infection developed febrile syndrome, and the other four presented with neurological disease: meningitis (n = 2), meningoencephalitis (n = 1) and severe depression of consciousness (n =1). These infections were most likely acquired in the districts of Faro (3), Lisbon (2) and Setúbal (1). In Portugal, the number of laboratory diagnostic requests for TOSV is low when compared to the numbers of requests for other less prevalent vector-borne viruses. The Faro district presented the highest number of TOSV-specific diagnostic requests which seems to indicate a higher level of recognition by clinicians in that region. Febrile syndrome and neurological disease were the clinical manifestations that were present in acute cases. In this study, in addition to the Faro district, recent infections were also detected in the districts of Lisbon and Setúbal. It is probable that TOSV may be distributed throughout the mainland territory since its main vector is present from north to south. In 2017, the sandfly fever Sicilian virus was associated for the first time with human disease in our country, thus alerting to the circulation of this phlebovirus. Even though the number of identified cases in Portugal is low, TOSV circulates and causes disease in our country. The diagnosis of this and other phleboviruses should not be neglected in the differential diagnosis of febrile syndrome and viral meningitis and meningoencephalitis, especially during the warmer months, when the vector's activity is higher.

Development and Assessment of Specific and High Sensitivity Reverse Transcription Nested Polymerase Chain Reaction Method for the Detection of <i>Aichivirus</i> A Monitoring in Groundwater

Development and Assessment of Specific and High Sensitivity Reverse Transcription Nested Polymerase Chain Reaction Method for the Detection of <i>Aichivirus</i> A Monitoring in Groundwater

Design and Evaluation of Multiplex One-Step Reverse Transcription PCR-Dipstick Chromatography Method for the Analysis of Seven Respiratory Pathogens.

Influenza A, influenza B, severe acute respiratory syndrome coronavirus 2, adenovirus, respiratory syncytial virus, Mycoplasma pneumoniae, and Chlamydophila pneumoniae are common pathogens that can cause severe pneumonia and other symptoms, resulting in acute lower respiratory tract infections. The objective of this study was to design and evaluate a sensitive and specific multiplex one-step reverse transcription PCR (RT-PCR)–dipstick chromatography method for simultaneous rapid detection of these seven pathogens. Streptavidin-coated blue latex particles were used to read out a positive signal. Based on the DNA–DNA hybridization of oligonucleotide sequences (Tag) for forward primer with the complementary oligonucleotide sequence (cTag) on the dipstick and biotin–streptavidin interactions, PCR products were able to be illuminated visually on the dipstick. The specificity and the limit of detection (LOD) were also evaluated. Moreover, the clinical performance of this method was compared with Sanger sequencing for 896 samples. No cross reaction with other pathogens was found, confirming the high specificity of this method. The LOD was 10 copies/µL for each of the tested pathogens, and the whole procedure took less than 40 min. Using 896 samples, the sensitivity and specificity were shown to be no lower than 94.5%. The positive predictive value was higher than 82.1%, and the negative predictive value was higher than 99.5%. The kappa value between the PCR–dipstick chromatography method and Sanger sequencing ranged from 0.869 to 0.940. In summary, our one-step RT-PCR–dipstick chromatography method is a sensitive and specific tool for rapidly detecting multiplex respiratory pathogens.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00284-021-02621-7.

RT-qPCR Assays for Rapid Detection of the N501Y, 69-70del, K417N, and E484K SARS-CoV-2 Mutations: A Screening Strategy to Identify Variants With Clinical Impact.

BackgroundSeveral variants of the SARS-CoV-2 have been documented globally during the current COVID-19 pandemic. The N501Y, 69-70del, K417N, and E484K SARS-CoV-2 mutations have been documented among the most relevant due to their potential pathogenic biological effects. This study aimed to design, validate, and propose a fast real-time RT-qPCR assay to detect SARS-CoV-2 mutations with possible clinical and epidemiological relevance in the Mexican population.MethodsTargeting spike (S) gene mutations of SARS-CoV-2 (N501Y, 69-70del, K417N, and E484K), specific primers, and probes for three specific quantitative reverse transcription PCR (RT-qPCR) assays were designed, and validated using Sanger sequencing. These assays were applied in clinical samples of 1060 COVID-19 patients from Jalisco Mexico.ResultsIn silico analyzes showed high specificity of the three assays. Amplicons of samples were confirmed through sequencing. The screening of samples of COVID-19 patients allowed the identification of the E484K mutation in nine individuals and the identification of P.2 Brazilian variant in Mexico.ConclusionThis work provides low-cost RT-qPCR assays for rapid screening and molecular surveillance of mutations with potential clinical impact. This strategy allowed the detection of E484K mutation and P.2 variant for the first time in samples from the Mexican population.

Epidemiological evaluation of sewage surveillance as a tool to detect the presence of COVID-19 cases in a low case load setting

In low prevalence settings the development of sensitive and specific quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) tests to detect SARS-CoV-2 (the virus causing COVID-19) in sewage presents the possibility of using sewage sampling as a diagnostic test for the presence of infected people in the catchment of the sampled sewer. However, the usefulness of such surveillance has not been quantified. In this study in the Australian state of Victoria between August and October 2020 the location of each known SARS-CoV-2-infected person was determined on each day from two days before onset to 55 days after, in 46 metropolitan and rural sewer catchments sampled weekly – a total of 71 positive and 275 negative samples, and 354,155 person-days of location data. These were categorised by time since onset and distance from the sampling site. The odds of detection in sewage were between 5 and 20 times higher where known cases were present, with less effect of distance than time since onset. Using positive qRT-PCR in a sewage sample as a diagnostic test not just for viral RNA in the sample, but for the presence of known infected people in the catchment on the same day, the sensitivity was moderate (31% to 76%) and the specificity high (87% to 94%). The odds of detection were increased with increased numbers of known infected people but decreased with increased distance and time since onset. The probability of detection of the viral subgenome in sewage samples was about 10% when one known infected person was present, and this increased with higher numbers of known infected people and greater proximity to the sampling site. Sewage surveillance can be used to detect people infected with SARS-CoV-2 in the catchment, directing a search for infectious clinical cases and other public health actions. However, detection at least eight weeks after onset may be due to existing cases rather than new ones, and, although not zero, the probability of detecting a single case is low.

Detecting Rapid Spread of SARS-CoV-2 Variants, France, January 26-February 16, 2021.

Variants of severe acute respiratory syndrome coronavirus 2 raise concerns regarding the control of coronavirus disease epidemics. We analyzed 40,000 specific reverse transcription PCR tests performed on positive samples during January 26–February 16, 2021, in France. We found high transmission advantage of variants and more advanced spread than anticipated.

Editorial: Coronavirus disease 2019 (COVID-19) - advances in epidemiology, diagnostics, treatments, host-directed therapies, pathogenesis, vaccines, and ongoing challenges.

Editorial: Coronavirus disease 2019 (COVID-19) - advances in epidemiology, diagnostics, treatments, host-directed therapies, pathogenesis, vaccines, and ongoing challenges.

Rapid and visual detection of the emerging novel duck reovirus by using a specific and sensitive reverse transcription recombinase polymerase amplification method

Duck spleen necrosis disease (DSND) caused by Novel Duck Reovirus (NDRV), is an emerging infectious disease that causes severely threaten to duck industry. Currently, the popular conventional RT-PCR technique for detecting NDRV is time consuming. So, it is essential to develop a rapid and accurate molecular diagnosis techniques of the pathogen for the purpose to effective control of the disease. In our study, a simple, rapid and reliable detection method was developed by an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA). The RT-RPA primers were designed targeting the S3 gene of NDRV, and its specificity was verified by testing a series of other waterfowl pathogens. A total of 20 field and experimental samples from infected ducklings were tested by the RT-RPA and compared with the results of the conventional RT-PCR and the quantitative RT-PCR simultaneously. The RT-RPA method could detect as little as 4.14 × 102 copies/μl of the target gene in the sensitivity analysis, which was 10×higher sensitive than the conventional RT-PCR. The major advantage of the RT-RPA method is that it could be performed as an isothermal reaction at 37 ℃ and completed within 20 min. In addition, no cross-reactivity was detected with other waterfowl-origin viruses. Also, the amplified products could be visualized faster, without the gel electrophoresis, by adding the SYBR Green I and observing them under an ultraviolet light. The newly developed RT-RPA method offers a simple, rapid and accurate for rapid detection of NDRV, which especially useful in on-site facilities and resource-limited areas.

Variability of cycle threshold values in an external quality assessment scheme for detection of the SARS-CoV-2 virus genome by RT-PCR.

The qualitative results of SARS-CoV-2 specific real-time reverse transcription (RT) PCR are used for initial diagnosis and follow-up of Covid-19 patients and asymptomatic virus carriers. However, clinical decision-making and health management policies often are based additionally on cycle threshold (Ct) values (i.e., quantitative results) to guide patient care, segregation and discharge management of individuals testing positive. Therefore, an analysis of inter-protocol variability is needed to assess the comparability of the quantitative results. Ct values reported in a SARS-CoV-2 virus genome detection external quality assessment challenge were analyzed. Three positive and two negative samples were distributed to participating test laboratories. Qualitative results (positive/negative) and quantitative results (Ct values) were assessed. A total of 66 laboratories participated, contributing results from 101 distinct test systems and reporting Ct values for a total of 92 different protocols. In all three positive samples, the means of the Ct values for the E-, N-, S-, RdRp-, and ORF1ab-genes varied by less than two cycles. However, 7.7% of reported results deviated by more than ±4.0 (maximum 18.0) cycles from the respective individual means. These larger deviations appear to be systematic errors. In an attempt to use PCR diagnostics beyond the identification of infected individuals, laboratories are frequently requested to report Ct values along with a qualitative result. This study highlights the limitations of interpreting Ct values from the various SARS-CoV genome detection protocols and suggests that standardization is necessary in the reporting of Ct values with respect to the target gene.

High-risk HPV infection modulates the promoter hypermethylation of APC, SFRP1, and PTEN in cervical cancer patients of North India.

Persistent infection with oncogenic HPV and downregulation of tumor suppressor genes play an essential role in the development and progression of cervical cancer. The present study aimed to identify the promoter methylation status of APC, SFRP1, and PTEN which are important regulators of Wnt pathway and their association with high-risk HPV infection and gene expression. Methylation Specific PCR (MSP) and quantitative reverse transcription PCR (RT-qPCR) were used to detect methylation status and gene expression levels of APC, SFRP1, and PTEN in cervical cancer biopsies (110) and paired non-cancerous biopsies (28). APC promoter was methylated in 38%, SFRP1 in 95%, and PTEN in 55% of the cervical cancer biopsies. Our data showed a trend of a higher rate of methylation of the gene promoters in cervical cancer biopsies while; they were majorly un-methylated in non-cancerous biopsies. Corresponding to a higher rate of methylation in cancer biopsies, the gene expression levels of APC, SFRP1, and PTEN were reduced in cervical cancer samples in comparison to normal cervix tissues. Further, we observed that 97% cancer biopsies were HPV infected and high-risk type HPV16 and 18 infections were significantly positively associated with APC (p = 0.008 and p = 0.007), SFRP1 (p = 0.003 and p = 0.0067), and PTEN (p = 0.049 and p = 0.008) promoter methylation. APC, SFRP1, and PTEN promoter hyper-methylation is positively associated with high-risk HPV infection and inversely associated with gene expression. Our findings show that high-risk HPV infection promotes methylation of these genes and further promotes their silencing.

Building the National SARS-CoV-2 Laboratory Diagnostic Capacity in Taiwan.

Through early and proactive laboratory testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes novel coronavirus 2019, Taiwan has demonstrated an efficient and rapid control response to contain the outbreak. Two days after the World Health Organization announced the complete viral genome sequence, the national laboratory of the Taiwan Centers for Disease Control developed a specific real-time reverse transcription polymerase chain reaction (real-time RT-PCR) test for SARS-CoV-2. The national laboratory network was further strengthened through the recruitment of medical centers and regional hospitals distributed throughout most geographical regions of the country. Ultimately, a network of 60 laboratories with a capacity of 7,342 real-time RT-PCR tests per day was established. Between January 14 and August 5, 2020, a total of 158,772 tests were conducted, corresponding to 120,487 cases. Test results were obtained within 24 hours, enabling an efficient and rapid control response.