Reverse Transcriptase-polymerase Chain Reaction Protocol Research Articles

Introducing a simple and cost-effective RT-PCR protocol for detection of DPYD*2A polymorphism: the first study in Kurdish population.

Fluoropyrimidines, the major chemotherapeutic agents in various malignancies treatment, are metabolized by dihydropyrimidine dehydrogenase (DPD). DPD deficiency can lead to severe and sometimes fatal toxicity. In the present study, we developed a simple protocol to detect the DPYD*2A variant. Common side effects in patients treated with these drugs were also evaluated in a Kurdish population. We established a reverse-transcriptase polymerase chain reaction (RT-PCR) technique for detection of DPYD*2A. Sanger sequencing was used to confirm the results. 121 Kurdish patients receiving fluoropyrimidine derivatives were enrolled, and clinical information regarding the dosage and toxicity was analyzed. Our RT-PCR method was able to detect one patient with heterozygous state for DPYD*2A (0.8%). The most observed adverse drug reactions were tingling, nausea, and hair loss. The frequency of patients with the toxicity of grade 3 or worse was 6.6%. This was the first study that detect DPYD*2A polymorphism in the Kurdish population. Our method was successfully able to detect the DPYD*2A variant and, due to its simplicity and cost-effectiveness, it may be considered as an alternative to the current methods, especially in developing countries. Our detected polymorphism rate at 0.8% is comparable with other studies. Despite the low rate of DPYD*2A polymorphism, pharmacogenetics assessment before beginning the treatment process is highly recommended due to its association with a high risk of severe toxicity.

Validation and standardization of designed N gene primer-based RT-PCR protocol for detecting Peste des Petits Ruminants virus in goats

The Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) test is one of the most popular and specific diagnostic tests to easily recognize the Peste des Petits Ruminants virus (PPRV) genome in clinical samples. The sensitivity of the RT-PCR test depends on gene-targeted primer sets. The literature appears to be lacking in designing primers used in RT-PCR to detect PPRV genome in Bangladesh. This study aimed to develop an N gene based PPRV primer set, a standardized RT-PCR protocol, and its validity test by comparison with other available primers. A total of seventy clinical samples and ten PPRV positive isolates were used in real-time RT-PCR and conventional RT-PCR using one pair designed primer set NF/NR and three pairs of published gene F1/F2, F1b/F2b and N1/N2. N gene-based PPRV primer sets (NF/NR) were designed from a published sequence of PPRV (Accession number GQ122187.1). Statistical analysis was carried out. The designed N gene-based primer positive PPRV samples were sequenced and analysed. The N gene-based primer sets were more sensitive to PPRV detection than F gene-based primer (P =.002) in the RT-PCR test. PPRV detects the highest (86%) of clinical samples in the RT-PCR test using a designed N gene-based primer. Sequence analysis showed that designed N gene-based the 402bp sequence of PPRV isolates is clustered with other Bangladeshi PPRV isolates and belongs to Lineage IV. New primers sets were designed from the conserved region of the N gene of PPRV. Designed primer sets successfully worked in real-time RT-PCR. The standardized RT-PCR protocol with the designed primer sets (NF/NR) can be used for the specific detection of PPRV from clinical samples.

Utility of Nucleic Acid Extraction Free COVID-19 Real Time PCR Protocol in Resource Limited Setting: A Pilot Study

Introduction: Coronavirus Disease (COVID-19), a respiratory infection, caused by severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), was first identified in Wuhan, Hubei province, China in December 2019. Alarming increase in the number of cases has put tremendous pressure on existing health resources. Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), a molecular diagnostic method, is considered gold standard for diagnosis of SARS-CoV-2 infection. It involves RNA extraction as the preliminary step. Innovations to cut down cost and time involved in SARS-CoV-2 testing are need of hour. Aim: The aim of this study was to assess the feasibility of Nucleic Acid Extraction Free (NEF) protocol for COVID-19 diagnosis in resource limited settings. Materials and Methods: In this pilot study a panel of 148 Nasopharyngeal (NP) samples was subjected to the novel NEF RT-PCR protocol and results were compared to gold standard RT-PCR on RNA extracted from NP specimen. The cycle threshold value for each target was tabulated in MS Excel Spreadsheet and data analysis was performed using Statistical Package for Social Sciences (SPSS) software version 15.0. Results: Out of 148 collected samples, 120 showed amplification of E and RdRp targets by RNA extraction-based RT-PCR. Overall sensitivity and specificity observed for NEF protocol was 43.94% and 96.42%, respectively. Conclusion: Further refinement in the protocol would be required to improve the sensitivity of NEF protocol and widespread use in laboratories.

Natural vertical infection by dengue virus serotype 4, Zika virus and Mayaro virus in Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus.

Vertical transmission to progeny ensures the maintenance of arboviruses in their natural vectors. This mechanism is largely reported for dengue virus (DENV) and yellow fever virus (YFV). Few studies have addressed this mechanism for Zika virus (ZIKV), Mayaro virus (MAYV) and other arboviruses. The present study investigated the natural infection rate by arboviruses in 4490 Aedes (Stegomyia) aegypti and 296 Aedes (Stegomyia) albopictus (Diptera: Culicidae) reared from eggs collected with ovitraps in Cuiabá, Mato Grosso State, from February to July, 2017. After viral RNA extraction and reverse transcriptase-polymerase chain reaction protocols for 10 flaviviruses and five alphaviruses, nucleotide sequencing and three passages in C6/36 cells, eight pools of Ae. aegypti positive for DENV-4 genotype II, seven for ZIKV Asian genotype and two for MAYV genotype L were found. In addition, two Ae. albopictus pools were positive for DENV-4 genotype II and two were positive for ZIKV Asian genotype. Infection was confirmed by viral isolation in all positive pools for DENV-4 and for MAYV and in eight of nine for ZIKV. This mechanism may contribute to the spread of arboviruses during epidemics and also to their maintenance in natural vectors during interepidemic periods.

The clinicopathology of foot and mouth disease and molecular epidemiology of the viruses circulated in cattle of Pabna district, Bangladesh

This study investigated outbreaks of Foot and Mouth Disease (FMD) in cattle in Pabna district, Bangladesh, during August-September 2015. Out of 100 cattle, 45 were infected with FMD virus, of which five young and two adult cattle died and the postmortem changes in internal organs were recorded. Oral tissue samples from infected cattle (n = 20) of four Upazillas (Sub-district) of Pabna district were collected for viral RNA extraction and serotype identification using reverse transcriptase polymerase chain reaction (RTPCR). Gross and histopathological changes in oral, pedal and mammary tissues were typical of FMD. Characteristics lesion of 'tiger heart disease' was seen in heart muscle of young and adult cattle. There was thickening of interlobular septum of lungs and characteristics of interstitial pneumonia. The uniplex and multiplex RT-PCR detected FMD viruses (430 bp) and FMD viral serotypes “O” (402 bp) and “Asia 1” (292 bp). Cattle of Sujanagar, Chatmahar and Isshardi Upazillas of Pabna district were infected with FMD viral serotype “O” and responsible for mortality of three young and two adult cattle. FMD viral serotype “Asia 1” causing death of two young cattle at Pabna Sadar. The fragment (485 bp) of Vp1 gene of FMD viral serotype “O” sequenced showed mutation in main antigenic sites. The phylogenetic analysis carried out with the Vp1 gene of FMD viral serotype “O” showed the viruses belonging to ME-SA topotype. The death of young and adult cattle was probably due to cardiac and/or respiratory failure. The adapted RTPCR protocol can be used in practice for detecting FMD viruses and its serotyping. Larger samples sizes require investigating to identify existing FMD viral serotypes and topotypes in order to design future preventive strategies.Bangl. vet. 2016. Vol. 33, No. 2, 39-50

Distribution of foot and mouth disease virus serotypes in cattle of Bangladesh

Foot and mouth disease (FMD) is a highly contagious viral infection in cloven hoofed domestic and wild animals and endemic in many countries of the world including Bangladesh. Clinical investigation was carried out to identify natural cases of FMD and characteristics signs of FMD like salivation, ulceration in oral and pedal tissues and lameness was seen. The specific serotypes of FMD viruses involved in infected cattle were, therefore, identified using reverse transcriptase polymerase chain reaction (RT-PCR). Samples (N=97) from oral lesions was collected from infected cattle from seven divisions of Bangladesh during May to December, 2013. Viral RNA was extracted from the infected oral tissues and FMD virus specific uniplex RT-PCR was designed to detect FMD viruses. Multiplex RT-PCR was adapted to detect serotype specific amplicons. Out of 97 samples tested in uniplex and multiplex RT-PCR, 92 and 90 samples showed amplification reaction for FMD virus and viral serotypes respectively. Among the 90 FMD virus specific positive identification, single infectivity due to FMD viral Serotypes O, A and Asia 1 were seen in 56 (62.2%), 13 (14.4%) and 16 (17.8%) cases respectively. Three cattle (3.3%) were co-infected with FMD viral Serotypes O and Asia 1 and two (2.2%) with FMD viral Serotypes O and A. FMD viral serotype O was dominating all over the country followed by Asia1 and A. Cattle of Bangladesh were infected with FMD viral serotype O, A and Asia 1 alone or in combination. The RT-PCR protocols designed and adapted successfully detected FMD viruses and viral serotypes in a fraction of the time required for virus isolation and serological detection. These RT-PCR protocols can be used for rapid serotyping of FMD viruses from filed infectivity and selection of vaccine viruses.SAARC J. Agri., 15(1): 33-42 (2017)

Time course of concurrent infection with dengue virus serotypes 2 and 4 detected in urine

Abstract Background In hyperendemic areas, concurrent multiserotype dengue virus (DENV) infections commonly occur in both humans and in mosquito vectors. Previous studies have focused on single blood specimens. Objectives To illustrate and characterize the time course of mixed infection with DENV serotypes 2 (DENV2) and 4 (DENV4) in a single case. Materials and methods Plasma, saliva, and urine were collected from a patient diagnosed with dengue hemorrhagic fever grade II and secondary DENV infection on days 7, 18, and 31 of his illness. DENV RNA detection was performed using 2 DENV-specific reverse transcriptase-polymerase chain reaction protocols. Cloning and sequencing were performed to quantify the major and minor viral populations in dual-serotype-infected specimen(s). Genotypes of both DENV serotypes were characterized. Results DENV genome was detected in plasma and saliva only at the first time point (day 7 of illness), but in urine at both the first and second time points (days 7 and 18 of illness). DENV4 serotype was found in all DENV genome-positive specimens. DENV2 serotype was unexpectedly also detected in the first urine specimen. DENV4 as the major and DENV2 as the minor population. The DENV2 serotype was clustered in genotype Asian I and DENV4 serotype in genotype I. Conclusion To our knowledge, this is the first report of concurrent multiserotype DENV infection detected solely in urine. Prospective noninvasive investigations may determine the prevalence of this phenomenon. Clinical and public health implications of this finding need to be explored.

Dengue virus in bats from southeastern Mexico.

To identify the relationship between landscape use and dengue virus (DENV) occurrence in bats, we investigated the presence of DENV from anthropogenically changed and unaltered landscapes in two Biosphere Reserves: Calakmul (Campeche) and Montes Azules (Chiapas) in southern Mexico. Spleen samples of 146 bats, belonging to 16 species, were tested for four DENV serotypes with standard reverse transcriptase polymerase chain reaction (RT-PCR) protocols. Six bats (4.1%) tested positive for DENV-2: four bats in Calakmul (two Glossophaga soricina, one Artibeus jamaicensis, and one A. lituratus) and two bats in Montes Azules (both A. lituratus). No effect of anthropogenic disturbance on the occurrence of DENV was detected; however, all three RT-PCR-positive bat species are considered abundant species in the Neotropics and well-adapted to disturbed habitats. To our knowledge, this study is the first study conducted in southeastern Mexico to identify DENV-2 in bats by a widely accepted RT-PCR protocol. The role that bats play on DENV's ecology remains undetermined.

A comprehensive analysis of breakpoint cluster region-abelson fusion oncogene splice variants in chronic myeloid leukemia and their correlation with disease biology.

BACKGROUND:BCR-ABL fusion oncogene is a hallmark of Chronic Myeloid Leukemia (CML). It results due to translocation between chromosome 22 and chromosome 9 [t (9; 22)(q34; q11)]. It gives rise to translation of a 210 KDa chimeric protein (p210), leading to enhanced tyrosine kinase activity and activation of leukemogenic pathways, ultimately causing onset of CML. In case of CML, the classic fusions are b2a2 or b3a2, fusing exon 13 (b2) or exon 14 (b3) of BCR, respectively, to exon 2 (a2) of ABL. The type of BCR-ABL transcripts are thought to be have different prognosis and hence useful in clinical decision-making. The frequencies of different fusion oncogenes associated with leukemia can vary in different ethnic groups and geographical regions due to interplay of genetic variation in different ethnic populations, diverse environmental factors and living style. Moreover, earlier relevant studies from our region were carried out in small subset of patients. Therefore, objective of this study was to find out frequencies of different BCR-ABL splice variants in larger subset of CML patients.METHODS:A nested reverse transcriptase polymerase chain reaction (RT-PCR) was established to detect BCR-ABL splice variants in 130 CML patients. Sensitivity of RT-PCR and ability to detect BCR-ABL fusion gene in least possible time was studied.RESULTS:BCR-ABL detection using our optimized RT-PCR protocol could be completed in 8 hours, starting from RNA extraction to Gel electrophoresis. Sensitivity of RT-PCR assay was of the order of 10−6. Out of 130 Pakistani patients, 83 (63.84%) expressed b3a2 while 47 (36.15%) expressed b2a2 transcript.CONCLUSION:Our RT-PCR was proved to be very quick to detect BCR-ABL fusion oncogene in CML patients within one working day. Because of its sensitivity, it can be used to monitor complete molecular response in CML. BCR-ABL RT-PCR and BCR-ABL splice variants frequency in our study differs from other ethnic groups. It shows that ethnic and geographical differences exist in BCR-ABL splice variant frequency, which may have a profound effect on disease biology as well as implications in prognosis and clinical management of BCR-ABL positive leukemias.

An improved one-tube RT-PCR protocol for analyzing single-cell gene expression in individual mammalian cells

It is well known that gene expression is regulated at the level of individual cells, and more evidence is now emerging that heterogeneity of cell regulation is orders of magnitude greater than previously thought. In order to detect meaningful variations in transcription levels, it is necessary to measure gene expression at single cell levels rather than in bulk cells, where individual differences or heterogeneity could be lost. In this work, we report an improved reverse-transcriptase polymerase chain reaction (RT-PCR) protocol which allows the direct measurement of gene expression in one tube (5-25 microl of total PCR mixture) at the single mammalian cell level. The protocol employs a new cell lysis buffer, and involves no RNA isolation or nested PCR steps, significantly reducing the possibility of contamination and errors. We successfully applied this protocol in qRT-PCR and linear-after-the-exponential (LATE)-PCR to analyze selected genes of various expression levels from three cell lines. Although further characterization of RNA stability is important, the preliminary results showed that gene expression heterogeneity could be common among members of genetically identical cell populations. The protocol illustrated can be utilized for a wide array of applications without much modification, such as cancer cell analysis and preimplantation genetic diagnostics. In addition, the protocol is based on intercalator-based (SYBR Green PCR) chemistry, which is less expensive and suitable for high-throughput platforms.

Polymerase chain reaction detection of Taura Syndrome Virus and infectious hypodermal and haematopoietic necrosis virus in frozen commodity tails ofPenaeus vannameiBoone

The prawn viruses White Spot Syndrome Virus and yellow head virus have been detected in frozen commodity prawns. However, no studies on the occurrence of two other Office International des Epizooties-listed viruses, Taura Syndrome Virus (TSV) or infectious hypodermal and haematopoietic necrosis virus (IHHNV), have been published. Therefore, a polymerase chain reaction (PCR) study on whether these two viruses could be found in frozen commodity prawns imported into Australia was undertaken. Amplicons indicative of TSV were found in 6/10 prawns collected at a retail outlet in Townsville and 10/10 prawns from Coff's Harbour using two different reverse transcriptase PCR protocols. Nucleotide sequences from the amplicons were between 97% and 99% similar to various TSV isolates from Asia. Amplicons indicative of the Philippine/American lineage of IHHNV were found in 5/5 and 5/6 prawns collected from Townsville and Coff's Harbour respectively. Clearly, frozen commodity Penaeus vannamei Boone from China can carry TSV and IHHNV with nucleic acid intact enough to be detected using PCR.

Effect of Preservative on Recoverable RT-PCR Amplicon Length from Influenza A Virus in Bird Feces

Surveillance for avian influenza A viruses in wild bird populations is often limited by requirements for a cold chain from time of specimen collection, by availability of ultra-low temperature specimen storage within a few hours or days, and by laborious classical virologic procedures. Successful storage of specimens in preservatives at ambient temperature and subsequent detection of RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) would assist in helping influenza surveillance efforts become more widespread in remote areas, as well as more timely and inexpensive. Here, we describe bird feces spiked with influenza A virus preserved with guanidine and commercial buffers or alcohols at ambient temperature and analyzed by RT-PCR protocols. Virus-specific RT-PCR products of, at most, 206 bp were recovered for samples preserved with alcohols and up to 521 bp for samples preserved with guanidine or commercial buffers. These results suggest that this approach is feasible in the field and that preserved specimens might be better assayed molecularly when preserved in guanidine or commercial buffers.

A Case of FIP1L1-PDGFRA-Positive Chronic Eosinophilic Leukemia with a Rare FIP1L1 Breakpoint

The idiopathic hypereosinophilic syndrome (HES) has remained for a long time a diagnosis of exclusion. Differential diagnosis between the HES and the related chronic eosinophilic leukemia (CEL) relied on the identification of signs of clonality that allowed, when present, the reclassification of patients as CEL. Recently, a new acquired mutation was described in approximately 50% of the HES/CEL patients: a cryptic deletion on chromosome band 4q12 generating a FIP1L1-PDGFRA fusion gene. According to the World Health Organization classification, this clonal abnormality has been proposed as a new surrogate marker for chronic eosinophilic leukemia diagnosis. Fluorescence in situ hybridization and reverse transcriptase-polymerase chain reaction protocols were developed for an accurate del(4)(q12q12) and FIP1L1-PDGFRA fusion gene detection. Here, we report a patient with a rare FIP1L1 intron 16 breakpoint located outside of the reported FIP1L1 breakpoint region (ie, from FIP1L1 introns 9 to 13). This case illustrates the risk of false-negative results with diagnostic procedures that do not take into account the occurrence of rare FIP1L1 breakpoints. As targeted therapy with tyrosine kinase inhibitors has dramatically changed the prognosis of FIP1L1-PDGFRA (+) CEL, false-negative results could hamper accurate diagnosis and treatment.

Optimization of Ribonucleic Acid Detection From Archival Guinea Pig Temporal Bone Specimens

The choice of ribonucleic acid (RNA) isolation protocol coupled with modifications to RNA extraction and detection procedures may result in a more reliable method to detect gene expression in archived temporal bones. A large number of archival temporal bones exist. Retrospective analysis of these specimens using techniques of RNA extraction will greatly enrich our understanding of the pathophysiology of specific otologic diseases. However, archival human temporal bones are aged and embedded in paraffin or celloidin, rendering isolation and manipulation of nucleic acid in preserved specimens difficult, especially as it pertains to RNA degradation. Despite some reports of moderate success in the recent past, RNA isolation and gene expression using polymerase chain reaction (PCR) analysis continues to be challenging and unreliable. Archival guinea pig temporal bone specimens were used to develop and optimize a protocol for RNA extraction and gene expression analysis using PCR and quantitative PCR methods. The genes amplified comprise housekeeping genes and genes associated with the glutamate pathway. Archival celloidin-embedded guinea pig temporal bones were collected from the senior author's collection of experimental hydropic inner ear specimens. RNA from this tissue was extracted using the protocol described previously in 16animals and using a modified trizol extraction technique in 10 animals. Gene expression analysis was performed on the extracted RNA. Analysis included two housekeeping genes, GAPDH and 18S, as well as three mediators of the glutamate pathway, glutamate aspartate transporter, glutamate synthetase, and inducible nitric oxide synthase. Compared with the standard extraction protocol, the trizol-based extraction technique showed greater reliability and reproducibility of RNA detection. The housekeeping gene GAPDH or 18S was detected in 7 of 36 attempts with the standard protocol versus 9 of 9 using the modified extraction method (P < 0.001). The gene of interest, glutamate aspartate transporter, was detected in 3 of 26 attempts with the standard protocol versus 12 of 13 attempts using the modified extraction method (P < 0.001). Quantification of messenger RNA levels was then achieved using quantitative PCR methods. Improved reliability for detection of gene expression and demonstration of reproducibility were accomplished by modification of RNA extraction technique and standard reverse transcriptase PCR protocol. In addition, we also showed that gene expression from archival material can be quantified by real-time PCR.

Simultaneous detection of Macrobrachium rosenbergii nodavirus and extra small virus by a single tube, one‐step multiplex RT‐PCR assay

Post-larvae of Macrobrachium rosenbergii infected with white tail disease were collected from hatcheries and nursery ponds in India. The causative organisms have been identified as Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). A one-step multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to detect these viruses simultaneously in naturally and experimentally infected prawns. Several parameters were assayed in order to optimize the protocol for simultaneous detection. Naturally and experimentally infected prawns showed two prominent bands of 681 and 500 bp for MrNV and XSV, respectively, as in separate RT-PCR assays. Experimentally infected adult prawns showed two bands for these two viruses in all the organs, except hepatopancreas and eyestalk, as seen in normal RT-PCR. The sensitivity test carried out on the primer sets of MrNV and XSV revealed that these primers could simultaneously detect the two viruses at a level of 25 fg of total RNA prepared from infected samples using this multiplex RT-PCR protocol.

Molecular diagnosis of paramyxovirus infection in snakes using reverse transcriptase-polymerase chain reaction and complementary deoxyribonucleic acid:ribonucleic acid in situ hybridization.

Identification of ophidian paramyxovirus (OPMV) nucleic acid was accomplished in 11 of 14 snakes by a reverse transcriptase-polymerase chain reaction (RT-PCR) assay that detected a 153-bp region of the OPMV genome in total RNA extracted from paraffin-embedded tissues and cell culture. The RT-PCR protocol amplified a portion of the OPMV RNA genome, producing a 153-bp complementary DNA (cDNA) product from both fresh and paraffin-embedded tissue samples. In addition, cDNA:RNA in situ hybridization localized OPMV in formalin-fixed, paraffin-embedded tissue specimens to specific tissues and cells. This latter technique increased the degree of specificity with which a diagnosis of OPMV could be made.

Studies on the occurrence of Macrobrachium rosenbergii nodavirus and extra small virus-like particles associated with white tail disease of M. rosenbergii in India by RT-PCR detection

A new disease similar to whitish tail disease (WTD) was observed in larvae and post-larvae of Macrobrachium rosenbergii in hatcheries and nursery ponds located at Nellore, Vijayawada and Chennai, India. Reverse transcriptase polymerase chain reaction (RT-PCR) assay on these samples revealed the presence of Macrobrachium rosenbergii nodavirus ( MrNV) and extra small virus (XSV) in infected larvae and post-larvae, as reported in China. Published primers and self-designed primers specific for MrNV were tested and compared for sensitivity with MrNV samples collected in India. Our designed primer sets 4 and 2 gave amplicons of 425 and 590 bp, respectively, with improved sensitivity to the level of 0.25 fg of total RNA when compared to 25 fg of total RNA for the previously published method. XSV detection was based on a recently published RT-PCR protocol. This is the first report on the occurrence of MrNV and XSV in WTD of freshwater prawn from India.

Quantification of mRNA for the vitamin D metabolizing enzymes CYP27B1 and CYP24 and vitamin D receptor in kidney using real-time reverse transcriptase- polymerase chain reaction.

Critical to an understanding of the control of 1,25-dihydroxyvitamin D (1,25D) activity is a molecular appreciation of the regulation of three genes, 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), 25-hydroxyvitamin D-24-hydroxylase (CYP24) and vitamin D receptor (VDR). We now report the sensitivity, reproducibility and accuracy of a real-time reverse transcriptase-polymerase chain reaction protocol (Taqman) for the quantification of mRNA levels for these genes in total RNA extracted from kidney tIssue. The sensitivity of the protocol was at least 150 copies of mRNA per reaction. Reproducibility, expressed as the coefficient of variation, ranged between 14 and 30% at the level of approximately 10(4) copies of mRNA per reaction. Accuracy was estimated at greater than 95% for each of these mRNAs. This protocol allows for the comparison of absolute mRNA levels in extracted total RNA in kidneys from animals fed diets containing different levels of calcium, ranging from 0.05% to 1%. Serum 1,25D levels were decreased when the dietary calcium concentration was increased (P<0.05). The levels of CYP27B1 mRNA were highest in the animals fed the 0.05% calcium diet (P<0.01). Conversely, CYP24 and VDR mRNA levels were highest in the animals fed the 1% calcium diet (P<0.01). Both CYP27B1 and CYP24 mRNA levels were major determinants of serum 1,25D levels when dietary calcium intakes were varied in these adult animals (Multiple R(2)=0.70, P<0.01). No significant relationship was detected between kidney CYP27B1 and serum parathyroid hormone (PTH) suggesting that serum calcium may regulate CYP27B1 mRNA expression directly during normocalcaemia. Low levels of CYP24 mRNA were associated with high PTH levels. These findings suggest that kidney CYP24 activity, possibly regulated by factors such as PTH, acts in concert with kidney CYP27B1 to control serum 1,25D levels.

Wilms tumor gene (WT1) expression as a panleukemic marker.

The Wilms tumor gene (WT1) is expressed in blasts of patients with acute leukemia, irrespective of lineage, and WT1 nuclear protein is detectable in the majority of such blasts. Only very few physiologic hematopoietic progenitors express WT1, but the WT1 expression level of these progenitors and that of leukemic blasts are comparable. Although not specific for acute hematologic malignant diseases, continuous WT1 expression in almost all leukemic blasts strikingly contrasts to its rather transient expression in very few physiologic hematopoietic progenitors. Quantitative and semiquantitative WT1 reverse transcriptase polymerase chain reaction (RT-PCR) protocols have limitations in discriminating physiologic from pathologic overall WT1 expression levels in mononuclear cell preparations. Because of these limitations, reports conflict on the usefulness of long-term monitoring of WT1 expression in patients with acute leukemia. Real-time quantitative WT1 RT-PCR protocols, however, have been developed and tested in small series of patients with acute leukemia. Such protocols hold promise to enable evaluation of the individual treatment response (short-term monitoring) and early diagnosis of imminent relapse through the detection and long-term monitoring of minimal residual disease in patients with acute leukemia. These protocols also should facilitate the notoriously difficult distinction between eosinophilic leukemia and hypereosinophilic syndromes. Data on WT1 expression in leukemic blasts and their physiologic counterparts are discussed in light of clinical relevance.

Enteroviruses detected by reverse transcriptase polymerase chain reaction from the coastal waters of Santa Monica Bay, California: low correlation to bacterial indicator levels.

Microbiological water quality at beaches is typically measured only by indicator bacteria, even though viruses are also a concern, because classical culture-based virus assays are not suitable. In this study, molecular-based assays for the detection of enteroviruses by reverse transcriptase polymerase chain reaction (RT-PCR) were performed on 50 coastal seawater samples taken from Santa Monica Bay, CA, over a six-year period, and compared with indicator bacteria. Sample sites were near freshwater outlets in the Bay and popular sandy beaches. RT-PCR is a primer-based molecular biology technique, used to detect the genomes of specific groups or types of viruses based upon conserved sequences. Results of the 50 analyses showed our ultrafiltration concentration methods and RT-PCR protocol could be used consistently to detect enteroviruses from 20 l samples of coastal seawater. Of the 50 samples, 16 (32%) were positive for enteroviruses, 27 (54%) were negative and 7 (14%) were inconclusive. There was no significant correlation between the presence of enteroviruses and individual standard microbiological indicators of fecal contamination, specifically total coliforms, fecal coliforms, or enterococci (r=0.14, r=0.28 and r=0.34, respectively, p>0.05). However, there was a significant correlation (r=0.71) to a combined set of bacterial water quality standards, involving all three indicators, recently adopted in California. There was no significant correlation between the RT-PCR results and levels of rainfall (a large source of runoff), but our analyses demonstrated that positive results for enteroviruses were significantly more likely during the winter `wet' season than during the summer `dry' season. Our results demonstrate that RT-PCR is an effective method for the detection of enteroviruses in coastal seawater, and they suggest that bacterial indicators are not necessarily good predictors of the presence of such viruses. Enteroviruses are known to be important etiological agents of disease from recreational water contact, so analysis for their presence might be advisable at certain locations (e.g. high-use sandy beaches) or during certain seasons of the year.