Real-time Reverse Transcription-polymerase Chain Reaction Method Research Articles

Detection of SARS-CoV-2 variants among travelers crossing the northern international border checkpoint in Duhok province, Iraq

Background: Variants of SARS-CoV-2 have been detected along with the worldwide COVID-19 transmission. Insufficient data exists regarding SARS-CoV-2 variants in Iraq, particularly concerning travelers at Iraqi border crossings. This study aimed to screen SARS-CoV-2 variants among individuals entering Iraq from Turkey through the Ibrahim Khalil international border checkpoint. Methods: A total of 116 (52 females, 64 males) COVID-19-positive cases from June 6, 2021, to July 30, 2021, were randomly selected from the Ibrahim Al-Khalil border crossing point between Turkey and Iraq. The patients were Iraqis tested positive for COVID-19 through nasopharyngeal and oropharyngeal swab collections. The confirmation of SARS-CoV-2 cases involved RNA extraction and real-time reverse transcription polymerase chain reaction (RT-PCR), following the World Health Organization guidelines. All samples were subjected to variant detection using the reverse transcription RT-PCR method. The RNA was extracted using a Zybio nucleic acid extraction kit and the RT-PCR was conducted using SARS-CoV-2 S-gene mutation detection RT-PCR kit. Results: In the 116 patients, we detected variants B.1.1.7, B.1.617.1.2, B.1.617.1.3, and Omicron sub-lineages (B.1.1.529, BA.1) at frequency rates of 13, 16, 45, and 36 cases, respectively. The highest prevalence rate was the delta variant among the tested subjects (38.8%) followed by the Omicron sub-lineages (B.1.1.529, BA.1) at 31%. Conclusion: This finding, which is the first of its kind at the northern Iraqi border checkpoint, suggests that these two variants could be a source of COVID-19 circulating in the Duhok province in Iraq. Additional studies are warranted across the country’s border entry points.

PERFORMANCE OF RAPID ANTIGEN TEST IN COMPARISON TO REAL-TIME RT-PCR IN DIAGNOSIS OF COVID-19 INFECTION: A SHORT STUDY

Objective: The global pandemic of Coronavirus Disease 2019 (COVID-19) is still spreading. Because of this, it is urgently necessary to develop quick, easy, and accurate assays to identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. For the purpose of diagnosing COVID-19 cases, the quick SARS-CoV-2 antigen detection test’s performance attributes should be assessed and contrasted with the gold standard real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. Methods: A retrospective analysis was done during January to May, 2022 at IPGME&R and SSKM Hospital, Kolkata, India. 2842 paired samples were taken from all patients reporting to the hospital for Rapid Antigen Test (RAT) and RT-PCR. The sensitivity and specificity were calculated to evaluate the performance of the RAT. Results: Of 2842 samples, 229 (8%) were positive, and 2613 (92%) were negative for SARS-CoV-2 RNA by real-time RT-PCR assay. The rapid SARS-CoV-2 antigen detection test’s sensitivity and specificity were 77.29% (177 out of 229) and 99.54% (2601 out of 2613), respectively. Positive predictive value was 93.65% and negative predictive value was 93% with the Ct value ≤35 of real-time RT-PCR result. Conclusion: RAT-positive samples are also positive by real-time RT-PCR test though RAT-negative patient with symptom, should further test for real-time RT-PCR as sensitivity is bit low particularly in symptomatic patient respect to real-time RT-PCR result. Hence, we can conclude that specificity- and sensitivity-wise real-time RT-PCR method is best, but during pandemic/outbreak situation, RAT is good alternative for fast screening and point-of-care test of COVID-19 detection.

Comparison of RT-PCR, RT-LAMP, and Antigen Quantification Assays for the Detection of SARS-CoV-2

A rapid and simple alternative test to real-time reverse transcription-polymerase chain reaction (RT-PCR) is required for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to help curb the spread of coronavirus disease (COVID-19). In the present study, we compared the RT-PCR method with chemiluminescent enzyme immunoassay (CLEIA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). We observed that the number of SARS-CoV-2 RNA copies and the CLEIA antigen quantification values were highly correlated. The detection limit for antigen quantification was 42.8 RNA copies for saliva samples and 23.4 copies for nasopharyngeal swab samples. For both purified RNA and purification-free crude RNA, the number of RNA copies and RT-LAMP threshold time (Tt) values were inversely correlated. RT-LAMP with purified RNA detected low copy numbers of RNA (5-50 copies), whereas fewer than 250 RNA copies could not be detected using crude RNA. CLEIA antigen quantification is potentially useful for large-scale screening, as it is compatible with high-throughput testing. RT-LAMP with crude RNA samples is applicable for rapid point-of-care testing because it can directly use patient specimens. It is important to select a diagnostic method that is simple and rapid when compared with RT-PCR, depending on the situation.

Comparison of a novel antigen detection test with reverse transcription polymerase chain reaction assay for laboratory diagnosis of SARS-CoV-2 infection.

Molecular diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse transcription polymerase chain reaction (RT-PCR) in respiratory specimens is considered the gold standard method. This method is highly sensitive and specific but it has some limitations such as being expensive and requiring special laboratory equipment and skilled personnel. RapidFor™ Antigen Rapid Test Kit is a commercially available Ag-RDT which is produced in Turkey and designed to detect the nucleocapsid antigen of SARS-CoV-2 in nasopharyngeal swab samples. The aim of this study was to evaluate the performance of this novel SARS-CoV-2 antigen detection considering the RT-PCR method as the gold standard. Four hundred forty-four nasopharyngeal swab samples which were collected from the patients who met clinical criteria of COVID-19 from ten centers in Turkey between September 2020 and February 2021 were included in the study. All the nasopharyngeal swab samples were tested for SARS-CoV-2 RNA using commercial RT-PCR kits (Bioeksen and A1 Lifesciences, İstanbul, Turkey) according to the manufacturer’s instructions. Viral loads were assessed according to the cycle threshold (Ct) values. RapidFor™ SARS-CoV-2 antigen test (Vitrosens Biotechnology, Istanbul, Turkey) was used to investigate the presence of SARS-CoV-2 antigen in all samples following the manufacturer’s instructions. Out of 444 nasopharyngeal swab samples tested, 346 (77.9%) were positive and 98 (22.1%) were negative for SARS-CoV-2 RNA by RTPCR. Overall sensitivity of the RapidFor™. Antigen Rapid Test Kit was 80.3% whereas specificity was found to be 87.8%. Positivity rate of rapid antigen test in samples with Ct values over 25 and below 30 was 82.7%, while it increased to 95.7% in samples 20 ≤ Ct < 25 and reached 100% in samples with Ct values below 20. RapidFor™ SARS-CoV-2 Ag test might be a good choice in the screening of symptomatic and asymptomatic patients and their contacts for taking isolation measures early, with advantages over RT-PCR as being rapid, easy and being applicable in every laboratory and even at point of care.

Comparative Evaluation of Cartridge-Based Abbott ID NOW Test With Probe-Based Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection of SARS-CoV-2.

Background: The gold standard test for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recommended by WHO is real-time reverse transcription polymerase chain reaction (RT-PCR), which has a turnaround time of five to six hours. Abbott ID NOW (Abbott Diagnostics Scarborough, Inc., Scarborough, ME, USA), the cartridge-based loop-mediated isothermal amplification (LAMP) assay, was approved by FDA for Emergency Use Authorization as rapid point of care testing. The present study was planned to evaluate the performance of the cartridge-based Abbott ID NOW test by comparing it to the currently used standard probe-based real-time RT-PCR method for detection of SARS-CoV-2.Methodology: A cross-sectional study was conducted in a tertiary care hospital in the eastern part of India after getting institutional ethics committee (IEC) approval. Two hundred fifty-nine cases of various age groups of both sexes who were advised for testing for SARS-CoV-2 were included in the study. Nasopharyngeal swabs were collected according to protocol advisory by the Indian Council of Medical Research (ICMR), India. Dry swabs were sent for Abbott ID NOW testing and swabs in viral transport medium were sent for probe-based RT-PCR assay using the CoviPath kit (Thermo Fisher Scientific, Bangalore, India). The data were collected and statistical analysis was performed using Statistical Package for Social Sciences (SPSS) (IBM Corp., Armonk, NY, USA). Sensitivity, specificity, positive and negative predictive values for ID NOW were calculated taking RT-PCR as the gold standard. Results: Out of 259 patients enrolled in the study, 49% were symptomatic for coronavirus disease 2019 (COVID-19). The prevalence rate of SARS-CoV-2 was 20.84% among the study population. Sensitivity and specificity, positive and negative predictive values of ID NOW test in comparison to RT-PCR assay was found to be 87%, 98%, 92.1% and 96.8% respectively. ID NOW detected seven out of 54 (12.9%) cases as false negative who were found to be positive with RT-PCR, with mean Ct value of the target genes >34.Conclusions: In this study the overall sensitivity for ID NOW assay was found to be lower, but specificity, positive and negative predictive values were found to be higher. It had the highest correlation to RT-PCR among symptomatic patients and at higher viral loads. Due to the ease of use and shortest result time for detecting COVID-19, ID NOW test could be used as a point-of-care test. But for all tests, the results should be interpreted according to the clinical and epidemiological context.

Hairpin-Spherical Nucleic Acids for Diagnosing COVID-19: a Simple Method to Generalize the Conventional PCR for Molecular Assays.

The COVID-19 pandemic revealed during the first global wave of this infectious disease that mass diagnostic testing was necessary to more rapidly detect infection in patients and control the pandemic. Therefore, extra research efforts to develop reliable and more accessible techniques for disease diagnosis are of supreme importance. Here, a target-responsive assembly of gold nanoparticle-core hairpin-spherical nucleic acids (AuNP-core H-SNAs) was implemented to modify the conventional polymerase chain reaction (PCR) assay for the “naked-eye” colorimetric detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Two hairpin DNA ligands are designed based on the two highly conserved regions within N and E genes of SARS-CoV-2 RNA by positioning two short palindromic arms (stem) on either side of a recognition sequence (loop). In the presence of a sequence-specific probe (activator), hairpin DNAs anchored to the surface of AuNPs unfold and expose the palindromic ends to the DNA-directed assembly of AuNPs. The sequence of the activator probes was chosen to be identical to the TaqMan probe in a real-time reverse transcription PCR (RT-PCR) assay for specifically targeting the N and E genes of SARS-CoV-2 RNA. They may either be degraded by the 5′-exonuclease activity of DNA polymerase during PCR cycles or stay intact depending on the presence or absence of the target template in the sample, respectively. Post-addition of H-SNA solutions to the final PCR products of some preconfirmed clinical samples for COVID-19 generated naked-eye-observable red and blue colors for positive and negative cases, respectively, with similar sensitivity to that of the real-time RT-PCR method.

Comparison of the Modified Centers for Disease Control and Prevention 2019-Novel Coronavirus Real-Time RT-PCR Method for Detection of Infectious and Heat-Inactivated Virus on Stainless Steel.

BackgroundInfectious Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was used in the validation of methods for detection of SARS-CoV-2 on stainless steel surfaces in the AOAC Research Institute Emergency Response Validation project. Handling infectious virus requires Biosafety Level (BSL)-3 facilities.ObjectiveTo compare the recovery and detection of infectious and heat-inactivated (65 °C for 30 min) SARS-CoV-2 from stainless steel by the modified US Centers for Disease Control and Prevention (CDC) 2019-Novel Coronavirus Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Diagnostic Panel.MethodsViral stocks were diluted in viral transport medium and deposited onto stainless steel test areas at 2 x 103 and 2 x 104 genomic copies for low and high, respectively. Test areas were sampled, and aliquots of the resulting test solutions analyzed by RT-qPCR according to the CDC method. Results were analyzed by Probability of Detection (POD) statistics.ResultsThe low level, where fractional positive results (25–75%) are expected, yielded PODI = 0.80 (0.58, 0.92) for the infectious virus and PODHI = 0.15 (0.05, 0.36) for the heat-inactivated virus. The bias, dPODHI = -0.65 (-0.80, -0.35), demonstrated a statistical difference between infectious and heat-inactivated virus detection. No difference was observed at the high inoculation level.ConclusionDespite the statistical difference observed, the use of the heat-inactivated virus is a viable alternative for matrix extension studies using a method comparison study design.HighlightsThe use of heat-inactivated SARS-CoV-2 can mitigate the need for a BSL-3 facility for matrix extension validation of alternative methods in SARS-CoV-2 studies.

Mechanism of maggot debridement therapy in promoting wound angiogenesis in patients with diabetic foot ulcer

Mechanism of maggot debridement therapy in promoting wound angiogenesis in patients with diabetic foot ulcer

Development of SYBR Green I-based real-time reverse transcription polymerase chain reaction for the detection of feline astrovirus

In this study, a SYBR Green I-based real-time reverse transcription-polymerase chain reaction (RT-PCR) was developed for the clinical diagnosis of feline astroviruses (FeAstVs). Specific primers were designed based on the conserved region of the FeAstV ORF1b gene. Experiments for specificity, sensitivity, and repeatability of the assay were carried out. In addition, the assay was evaluated using clinical samples. Specificity analysis indicated that the assay showed negative results with samples of Feline Parvovirus, Feline Herpesvirus, Feline Calicivirus, Feline Bocavirus, and Feline Coronavirus, indicating good specificity of the assay. Sensitivity analysis showed that the SYBR Green I-based real-time RT-PCR method could detect as low as 3.72 × 101 copies/μL of template, which is 100-fold more sensitive compared to the conventional RT-PCR. Both intra-assay and inter-assay variability were lower than 1 %, indicating good reproducibility. Furthermore, an analysis of 150 fecal samples showed that the positive detection rate of SYBR Green I-based real-time RT-PCR was higher than that of the conventional RT-PCR, indicating the high reliability of the method. The assay is cheap and effective. Therefore, it could provide support for the detection of FeAstV in large-scale clinical testing and epidemiological investigation.

Low prevalence of IgG antibodies to SARS‐CoV‐2 in cancer patients with COVID‐19

Low prevalence of <scp>IgG</scp> antibodies to <scp>SARS‐CoV</scp>‐2 in cancer patients with <scp>COVID</scp>‐19

An Ultra-Rapid Real-Time RT-PCR Method Using PCR1100 for Detecting Human Orthopneumovirus.

Human orthopneumovirus, also known as the respiratory syncytial virus (RSV), is a leading cause of respiratory tract infections in children worldwide. The World Health Organization has taken steps toward establishing a global surveillance system for RSV, based on the global influenza surveillance and response system initiated in 2015. The US Centers for Disease Control and Prevention (CDC) has developed a genetic detection method based on real-time reverse transcription polymerase chain reaction (RT-PCR), which is used in global RSV surveillance. In Japan, immunoassay-based rapid antigen detection kits are widely used for the detection of RSV. In this study, an ultra-rapid real-time RT-PCR method for the rapid detection of RSV was developed using the PCR1100 device based on the US CDC assay in order to detect RSV in comparable time to rapid test kits. The ultra-rapid real-time RT-PCR could detect RSV viral RNA in less than 20 min while maintaining sensitivity and specificity comparable to conventional real-time RT-PCR using large installed instruments. Furthermore, combining ultra-rapid real-time RT-PCR with the M1 Sample Prep kit reduced the total working time for the detection of RSV from clinical specimen to less than 25 min, suggesting this method could be used for point-of-care RSV testing.

Investigation and successful control of an echovirus 11 outbreak in neonatal intensive care units

Echovirus 11 emerged as a predominant enterovirus strain and was associated with neonatal mortalities in Taiwan in 2018. We investigated an echovirus 11 outbreak in the neonatal intensive care units (NICUs) in a tertiary hospital in northern Taiwan and analyzed infection control efforts. Between May and June 2018, an outbreak of 10 infants with echovirus 11 infections occurred in the NICUs. Comprehensive surveillance, including virus isolation, real-time reverse transcription-polymerase chain reaction (RT-PCR), and consequential degenerate hybrid oligonucleotide primer (CODEHOP) methods, were arranged for specimens (rectal or throat swabs), which were obtained from all contacts, newly admitted cases, and suspected cases during the outbreak since June 2. Ten cases were identified with echovirus 11 infection in this outbreak. Eight of these 10 confirmed cases were identified by viral isolation, and the remaining two cases were identified by RT-PCR surveillance. In addition to confirmed cases, the surveillance of 19 contacts, 47 newly admitted cases, and nine suspected cases showed negative results. All confirmed cases eventually recovered. RT-PCR and CODEHOP methods significantly shorten the time of laboratory diagnosis of enterovirus infection compared with conventional methods. The outbreak of echovirus 11 in the NICUs was caused by three imported cases and was successfully controlled by the implementation of isolation, rapid surveillance, reinforced disinfection, and infection control measures.

Memory Function, Neurological, and Immunological Biomarkers in Allergic Asthmatic Mice Intratracheally Exposed to Bisphenol A.

Bisphenol A (BPA) is a major constituent of plastic products, including epoxy resin containers, mobile phones, dental sealants, as well as electronic and medical equipment. BPA is recognized as an endocrine system-disrupting chemical which has toxic effects on the brain and reproductive system. However, little is known about the effects of co-exposure of BPA with allergens on the memory function and neurological as well as immunological biomarker levels. In this study, we examined the effects of intratracheal instillation of BPA on the memory function and neuroimmune biomarker levels using a mouse model of allergic asthma. Male C3H/HeJ Jcl mice were given three doses of BPA (0.0625 pmol, 1.25 pmol, and 25 pmol BPA/animal) intratracheally once a week, and ovalbumin (OVA) intratracheally every other week from 5 to 11 weeks old. At 11 weeks of age, a novel object recognition test was conducted after the final administration of OVA, and the hippocampi and hypothalami of the animals were collected after 24 h. The expression levels of the memory function-related genes N-methyl-D-aspartate (NMDA) receptor subunits, inflammatory cytokines, microglia markers, estrogen receptor-alpha, and oxytocin receptor were examined by real-time RT-PCR (real-time reverse transcription polymerase chain reaction) and immunohistochemical methods. Impairment of the novel object recognition ability was observed in the high-dose BPA-exposed mice with allergic asthma. In addition, the allergic asthmatic mice also showed downregulation of neurological biomarkers, such as NMDA receptor subunit NR2B in the hippocampus but no significant effect on immunological biomarkers in the hypothalamus. These findings suggest that exposure to high-dose BPA triggered impairment of memory function in the allergic asthmatic mice. This is the first study to show that, in the presence of allergens, exposure to high-dose BPA may affect memory by modulating the memory function-related genes in the hippocampus.

Development of real-time RT-PCR assays for detection of three classes of HHV-6A gene transcripts.

Human herpesvirus 6 (HHV-6), a member of the betaherpesvirus family, has two distinct species: HHV-6A and HHV-6B. HHV-6B real-time reverse transcription polymerase chain reaction (RT-PCR) has been used to distinguish between active and latent viral infection. In this study, we developed a real-time RT-PCR assay to detect HHV-6A-specific transcripts and evaluated its reliability for analysis of clinical samples. To develop HHV-6A-specific real-time RT-PCR assays, three different classes of gene transcripts (immediate early: U90; early: U12; and late: U100) were selected as targets. Serial d ilutions of plasmid DNAs containing target sequences and RNAs extracted from HHV-6A-infected cells were used to determine assay specificity and sensitivity. Peripheral blood mononuclear cells (PBMCs) collected from patients with either primary or reactivated HHV-6B infection, and one patient with X-linked severe combined immunodeficiency (X-SCID) with HHV-6A reactivation, were used to evaluate assay reliability. The HHV-6A-specific real-time RT-PCR assays amplified plasmids containing the target sequences at concentrations between 10 and 1 × 106 copies per reaction. The intra-assay coefficients of variation were less than 5%. The three classes of HHV-6A gene transcripts were not detected in any HHV-6B sample isolated from the patients. In the X-SCID patient, high copy numbers of HHV-6A U12 and U100 transcripts were detected in PBMC samples during viremia. Thus, we successfully established highly sensitive and reproducible real-time RT-PCR methods targeting three classes of HHV-6A gene transcripts. This method should be useful for discriminating active HHV-6A infection from either latent infection or chromosomally integrated HHV-6A (ciHHV-6A).

Development of a comprehensive detection and identification molecular based system for eight pospiviroids

Pospiviroids cause serious economic damage to solanaceous crops, including tomato (Solanum lycopersicum), and cause symptomless infections in many horticultural plants. Symptomless infection and seed transmission are obstacles in visual inspection for plant quarantine. Therefore, in the present study, a comprehensive detection and identification system for molecular diagnosis of eight pospiviroids was developed, based on real-time reverse transcription polymerase chain reaction (RT-PCR). In this system, two real-time RT-PCR methods based on SYBR Green and TaqMan technologies were combined. First, tomato seed samples were screened for all the eight pospiviroids by the SYBR Green method using three primer pairs, including one pair of universal primers. Samples that were positive using the universal primer set were subjected to secondary screening for the identification to species level of six viroids by TaqMan real-time RT-PCR method, with specific primer and probe sets. This two-phase system could specifically detect a viroid in a sample of 400 seeds containing a single contaminated seed. The developed system could be an effective and simple tool to avoid the introduction of pospiviroids through tomato seeds.

Molecular characterization of the coat protein gene of prunus necrotic ringspot virus infecting peach in Montenegro

In 2011 and 2012, two major peach-growing areas in Montenegro were surveyed for the occurrence of eleven peach viruses. Samples collected from 105 peach and nectarine trees were tested by Reverse transcription-polymerase chain reaction (RT-PCR) using sets of primers specific for the detection of Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), Cherry mottle leaf virus (CMLV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Peach mosaic virus (PMV), Peach rosette mosaic virus (PRMV), Tobacco ringspot virus (TRSV) and Tomato ringspot virus (ToRSV). The occurrence of Strawberry latent ringspot virus (SLRSV) was examined using nested RT-PCR, while a Real-time quantitative RT-PCR method was applied for detection of Plum pox virus (PPV). PPV, PNRSV and PDV were detected in 42.9, 24.8 and 0.9 % of samples. Mixed viral infections were recorded in 6.7 % of samples, while results showed the absence of ACLSV, ApMV, PMV, CMLV, SLRSV, PRMV, TRSV and ToRSV from all assayed samples. In particular, the PCR products of complete coat protein (CP) gene was cloned and sequenced from fourteen PNRSV isolates. A high identity (87.5–100 %) was found between CP nucleotide sequences of Montenegrin isolates and other PNRSV isolates from different hosts and geographic regions retrieved from GenBank. CP nucleotide sequence comparison, as well as phylogenetic reconstruction confirmed the clustering of the isolates into three molecular groups (PV-96, PV-32 and PE-5), whereby Montenegrin isolates were separated into two phylogroups (PV-96 and PE-5). Comparative sequence and phylogenetic analysis showed that clustering of various isolates was not associated with geographic and host origin.

Identification and validation of reference genes for gene expression studies in postharvest rose flower (Rosa hybrida)

Optimal reference genes are important for data normalization so that accurate and reliable gene expression measurements may be obtained in both semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR) methods. This study firstly evaluated potential reference genes in petals of rose flower (Rosa hybrida) under postharvest stress conditions and in various floral organs during flower opening and senescence, combining both RT-PCR and qRT-PCR analysis. The expression stabilities of gene members from three traditional housekeeping gene families – actin (RhACT), tubulin (RhTUB) and ubiquitin (RhUBI) – were assessed using two analysis software packages, geNorm and NormFinder. The results showed that, for cut rose flower, the optimal reference genes were RhUBI1 for dehydration treatment and receptacles; RhTUB2 for exogenous ethylene; RhACT4 for gibberellic and abscisic acid treatments, wounding and stamens; RhUBI6 for petals; RhUBI2 for sepals; and RhACT1 for gynoecia, respectively. Our results provide guidelines for reference gene(s) selection under different postharvest conditions and point the way towards more accurate and widespread use of qRT-PCR in rose flower.

Application of real-time reverse transcription polymerase chain reaction to the detection the matrix, H5 and H7 genes of avian influenza viruses in field samples from South Korea

BackgroundThe rapid and accurate identification of the H5 and H7 subtypes of avian influenza (AI) virus is an important step for the control and eradication of highly pathogenic AI outbreaks and for the surveillance of AI viruses that have the potential to undergo changes in pathogenicity in poultry and wild birds. Currently, real-time reverse transcription polymerase chain reaction (RRT-PCR) is routinely used for the rapid detection of the H5 and H7 genes, but misidentification is frequent for emergent isolates and viruses isolated from diverse regions due to the high sequence variation among AI viruses.FindingsIn this study, an RRT-PCR method was tested for the detection of matrix, H5 and H7 genes from diverse subtypes of AI viruses and from field samples obtained through AI surveillance in South Korea over the last four years. Both RRT-PCR and conventional experiment (virus isolation using egg inoculation followed by reverse transcription polymerase chain reaction) agreed on the virus-positive samples. And the comparison of the results with 174 clinical samples showed a high level of agreement without decreasing the specificity and sensitivity.ConclusionsThis assay could be useful tool for the rapid detection of AI using the field samples from domestic poultry and wild birds in South Korea, and continuous regional updates is needed to validate primer sets as the AI virus evolves.

Prognostic significance of CTCs and CSCs of tumor drainage vein blood in Dukes' stage B and C colorectal cancer patients

The clinical significance of circulating tumor cells (CTCs) including cancer stem cells (CSCs) (CTC/CSC) in the tumor drainage vein blood of patients with colorectal cancer (CRC) is unclear. In this study, we investigated the prognostic value of CTC/CSC that express carcinoembryonic antigen (CEA) cytokeratin 19 (CK19), CK20 and/or CD133 (CEA/CK/CD133) mRNA in the tumor drainage blood of CRC patients with Dukes' stage B and C. We examined tumor drainage blood from 197 patients with Dukes' stage B and C CRC. CTCs that expressed CEA, CK19, CK20 and CD133 mRNA were detected using the quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. Each mRNA level was normalized with GAPDH mRNA levels. In the relationship between the expression of CEA/CK/CD133 in the tumor drainage blood and clinicopathological factors, a significant correlation was observed between CEA/CK/CD133 expression and Dukes' stage (p<0.041). In CRC patients with Dukes' stage B and C, disease-free (DFS) and overall survival (OS) of patients with CEA/CK/CD133 positive in the tumor drainage blood were significantly worse than that of marker gene negative patients. In contrast, in patients with Dukes' stage A, no significant differences were shown between these groups. By Cox progression analysis, it was shown that CEA/CK/CD133 mRNA in tumor drainage blood was an independent prognostic factor for DFS and OS in patients with Dukes' stage B and C. These results suggest that detecting CEA/CK/CD133 mRNA in tumor drainage blood by the real-time RT-PCR method would have a prognostic value in CRC patients with Dukes' stage B and C.

Quantitative Analysis of UGT2B28 mRNA Expression by Real-Time RT-PCR and Application to Human Tissue Distribution Study

We recently established a method for quantitative determination of human catalytically active UDP-glucuronosyltransferases (UGTs) other than UGT2A1 and UGT2B28 by real-time reverse transcription-polymerase chain reaction (RT-PCR), and applied the method to an exhaustive analysis of localization in various human tissues. We report here an additional quantitative determination method targeting UGT2B28. To date, there have been no reports on the distribution of UGT2B28 mRNA expression in human tissues based on quantitative determination. Human UGT2B28 was clearly detected in the breast and adipose tissue. UGT2B28 expression in the breast was comparatively low, about 1.6% of GAPDH mRNA levels, and was less than 5% of normalized (against GAPDH) UGT2B7 and 2B10 mRNA expression levels in the liver. Although the UGT2B28 has 97% identity with UGT2B11 at the cDNA sequence level, the primers constructed for UGT2B28 did not detect UGT2B11. In addition, significant expression of UGT2B11 was detected in the liver, breast and kidney, and was clearly different from the distribution of UGT2B28. Therefore, we conclude that the real-time RT-PCR method established here is very specific for human UGT2B28 isozyme.