Real-time Polymerase Chain Reaction Primers Research Articles

Rapid Diagnostic Testing for Response to the Monkeypox Outbreak - Laboratory Response Network, United States, May 17-June 30, 2022.

As part of public health preparedness for infectious disease threats, CDC collaborates with other U.S. public health officials to ensure that the Laboratory Response Network (LRN) has diagnostic tools to detect Orthopoxviruses, the genus that includes Variola virus, the causative agent of smallpox. LRN is a network of state and local public health, federal, U.S. Department of Defense (DOD), veterinary, food, and environmental testing laboratories. CDC developed, and the Food and Drug Administration (FDA) granted 510(k) clearance* for the Non-variola Orthopoxvirus Real-time PCR Primer and Probe Set (non-variola Orthopoxvirus [NVO] assay), a polymerase chain reaction (PCR) diagnostic test to detect NVO. On May 17, 2022, CDC was contacted by the Massachusetts Department of Public Health (DPH) regarding a suspected case of monkeypox, a disease caused by the Orthopoxvirus Monkeypox virus. Specimens were collected and tested by the Massachusetts DPH public health laboratory with LRN testing capability using the NVO assay. Nationwide, 68 LRN laboratories had capacity to test approximately 8,000 NVO tests per week during June. During May 17-June 30, LRN laboratories tested 2,009 specimens from suspected monkeypox cases. Among those, 730 (36.3%) specimens from 395 patients were positive for NVO. NVO-positive specimens from 159 persons were confirmed by CDC to be monkeypox; final characterization is pending for 236. Prompt identification of persons with infection allowed rapid response to the outbreak, including isolation and treatment of patients, administration of vaccines, and other public health action. To further facilitate access to testing and increase convenience for providers and patients by using existing provider-laboratory relationships, CDC and LRN are supporting five large commercial laboratories with a national footprint (Aegis Science, LabCorp, Mayo Clinic Laboratories, Quest Diagnostics, and Sonic Healthcare) to establish NVO testing capacity of 10,000 specimens per week per laboratory. On July 6, 2022, the first commercial laboratory began accepting specimens for NVO testing based on clinician orders.

Vitellogenin Gene Expression and Sex Steroid Levels as Biomarkers in Yellowfin Seabream (Acanthopagrus latus) Exposed to Bisphenol-A

Background: The egg yolk precursor protein vitellogenin (VTG) has proven to be a useful biomarker, used to identify organisms exposed to estrogenic compounds. Methods: We investigated variations in the VTG gene expression pattern and plasma sex steroid hormones concentrations in the yellowfin Seabream, Acanthopagrus latus, (A. latus) by various doses of bisphenol-A (BPA) exposure for 7 and 14 days. We developed a quantitative real time polymerase chain reaction (RT-PCR) assay for the expression of VTG gene in A. latus. The dose-response pattern of VTG gene expression in A. latus exposed to various doses of BPA was characterized. In order to design RT-PCR primers specific to A. latus VTG, a partial sequence of the VTG gene was obtained. Results: The RT-PCR assay was effective in detecting increased VTG gene expression in A. latus exposed to BPA. It also demonstrated that the VTG expression was affected by BPA in a dose and time-dependent manner. Plasma testosterone (T) levels were decreased in the treated fish in comparison with those found in the control group, when they were exposed to 100 µg/g of BPA and 2 µg/g of E2. In contrast, the plasma levels of 17β-estradiol (E2) were significantly increased in a dose-dependent manner. Conclusion: The results suggest that VTG mRNA quantification can provide a sensitive and early signal in the detection of estrogens in marine wildlife. It also indicated that BPA could lead to an imbalance of sex steroid hormones with potentially harmful consequences on sexually immature male A. latus.

LDOC1 regulates Wnt5a expression and osteosarcoma cell metastasis and is correlated with the survival of osteosarcoma patients.

Osteosarcomas are common bone malignancies in children and adolescents. LDOC1 (leucine zipper, down-regulated in cancer 1), a tumor suppressor, is down-regulated in many cancers. In this study, we investigated the role of LDOC1 in tumor metastasis and its prognostic significance in osteosarcomas. We established osteosarcoma cells stably expressing LDOC1, driven by an HIV-based lentiviral system. We investigated the impact of LDOC1 on migration and invasion abilities in these cells using a transwell assay. LDOC1-associated changes in expression of metastasis-promoting genes were analyzed with a quantitative real-time polymerase chain reaction primer array. A xenograft tumor model (n = 7 mice/group) was used to assess the effect of LDOC1 on osteosarcoma metastasis in vivo. The overall survival and disease-free survival of osteosarcoma patients (n = 74) were analyzed retrospectively based on immunohistochemical analysis of LDOC1 levels in tumors and Kaplan-Meier analysis. LDOC1-expressing osteosarcoma cells displayed decreased migration and invasion in vitro. The quantitative real-time polymerase chain reaction primer array data showed that increased LDOC1 expression up-regulated many metastasis-suppressor genes. In the xenograft model, micro-computed tomography imaging data indicated that increased LDOC1 expression is associated with weaker lung metastasis ability. The Wnt5a signaling pathway promotes osteosarcoma metastasis; LDOC1 expression decreased Wnt5a levels in osteosarcoma cells. Kaplan-Meier analysis showed that higher LDOC1 expression was associated with improved osteosarcoma patient overall survival and disease free survival (p = 0.022). Our data show that LDOC1 is a tumor suppressor in osteosarcoma, and that it regulates metastasis of osteosarcoma cells. Furthermore, LDOC1 might be a valuable prognostic marker in osteosarcomas.

Analyzing the gene expression profile of anaplastic histology Wilms' tumor with real-time polymerase chain reaction arrays.

BackgroundWilms’ tumor (WT) is one of the most common malignant neoplasms of the urinary tract in children. Anaplastic histology (unfavorable histology) accounts for about 10% of whole WTs, and it is the single most important histologic predictor of treatment response and survival in patients with WT; however, until now the molecular basis of this phenotype is not very clearly.MethodsA real-time polymerase chain reaction (PCR) array was designed and tested. Next, the gene expression profile of pediatric anaplastic histology WT and normal adjacent tissues were analyzed. These expression data were anlyzed with Multi Experiment View (MEV) cluster software further. Datasets representing genes with altered expression profiles derived from cluster analyses were imported into the Ingenuity Pathway Analysis Tool (IPA).Results88 real-time PCR primer pairs for quantitative gene expression analysis of key genes involved in pediatric anaplastic histology WT were designed and tested. The gene expression profile of pediatric anaplastic histology WT is significantly different from adjacent normal controls; we identified 15 genes that are up-regulated and 16 genes that are down-regulated in the former. To investigate biological interactions of these differently regulated genes, datasets representing genes with altered expression profiles were imported into the IPA for further analysis, which revealed three significant networks: Cancer, Hematological Disease, and Gene Expression, which included 27 focus molecules and a significance score of 43. The IPA analysis also grouped the differentially expressed genes into biological mechanisms related to Cell Death and Survival 1.15E−12, Cellular Development 2.84E−11, Cellular Growth and Proliferation 2.84E-11, Gene Expression 4.43E−10, and DNA Replication, Recombination, and Repair 1.39E−07. The important upstream regulators of pediatric anaplastic histology WT were TP53 and TGFβ1 signaling (P = 1.15E−14 and 3.79E−13, respectively).ConclusionsOur study demonstrates that the gene expression profile of pediatric anaplastic histology WT is significantly different from adjacent normal tissues with real-time PCR array. We identified some genes that are dysregulated in pediatric anaplastic histology WT for the first time, such as HDAC7, and IPA analysis showed the most important pathways for pediatric anaplastic histology WT are TP53 and TGFβ1 signaling. This work may provide new clues into the molecular mechanisms behind pediatric anaplastic histology WT.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-015-0197-x) contains supplementary material, which is available to authorized users.

Absence of lysogeny in wild populations of Erwinia amylovora and Pantoea agglomerans.

Lytic bacteriophages are in development as biological control agents for the prevention of fire blight disease caused by Erwinia amylovora. Temperate phages should be excluded as biologicals since lysogeny produces the dual risks of host resistance to phage attack and the transduction of virulence determinants between bacteria. The extent of lysogeny was estimated in wild populations of E. amylovora and Pantoea agglomerans with real–time polymerase chain reaction primers developed to detect E. amylovora phages belonging to the Myoviridae and Podoviridae families. Pantoea agglomerans, an orchard epiphyte, is easily infected by Erwinia spp. phages, and it serves as a carrier in the development of the phage-mediated biological control agent. Screening of 161 E. amylovora isolates from 16 distinct geographical areas in North America, Europe, North Africa and New Zealand and 82 P. agglomerans isolates from southern Ontario, Canada showed that none possessed prophage. Unstable phage resistant clones or lysogens were produced under laboratory conditions. Additionally, a stable lysogen was recovered from infection of bacterial isolate Ea110R with Podoviridae phage ΦEa35-20. These laboratory observations suggested that while lysogeny is possible in E. amylovora, it is rare or absent in natural populations, and there is a minimal risk associated with lysogenic conversion and transduction by Erwinia spp. phages.

Establishment of a CYP2C19 Genotyping Assay for Clinical Use

Conversion of clopidogrel (Plavix) to its active metabolite is catalyzed largely by the P450 enzyme 2C19 (CYP2C19). Numerous allelic variants of CYP2C19 exist. The *1 allele is considered wild type, whereas the *2 and *3 alleles have no in vivo enzymatic activity. Conversely, the *17 allele has increased expression, resulting in increased clopidogrel activation. Poor metabolizers (*2/*2 and *2/*3 genotypes) experience higher rates of therapeutic failure. For this reason, we have validated a CYP2C19 genotyping assay for the *1, *2, *3, and *17 alleles. Genomic DNA extracted from 30 deidentified EDTA whole-blood samples from patients was analyzed at 2 independent facilities using specific TaqMan realtime polymerase chain reaction primers and probes. Concordant genotypes were generated on all samples tested. Of the 30 samples, 15 were CYP2C19*1/*1, 8 were CYP2C19*1/*17, 5 were CYP2C19*1/*2, and 2 were CYP2C19*2/*17. There were no CYP2C19*3 alleles or *2/*2 homozygous genotypes detected. This CYP2C19 genotyping assay is appropriate for clinical testing, demonstrating excellent interlaboratory concordance, enabling the selection of the most effective clopidogrel treatment regimen for patients undergoing percutaneous coronary intervention.

Quantitative analysis of microorganism composition in a pilot-scale fermentative biohydrogen production system

Five specific real-time polymerase chain reaction primers targeting the 16S rRNA gene of Clostridium spp., Klebsiella spp., Streptococcus spp., Pseudomonas spp., and Bifidobacterium spp., and two primer sets targeting the hydrogenase genes of hydrogen-producing Clostridium pasteurianum and Clostridium butyricum were designed and tested in the present study to quantify the microorganisms in fermentative biohydrogen production systems. The former primers revealed the composition of all coexisting microorganisms, whereas the latter ones provided information on which clostridia were responsible for the biohydrogen production in various operational conditions. When sucrose was selected as the feeding substrate, the biogas production and hydrogen production rate (HPR) of the system increased as the percentage of Clostridium spp. (especially C. pasteurianum) increased. The cell count of C. pasteurianum increased up to 90% of the total cell population when the system approached its maximum hydrogen production. C. butyricum was identified as the main hydrogen-producing clostridium in the condensed molasses soluble wastewater feeding system, but there was no significant correlation between system HPR and C. butyricum cell count. At the same time, other microorganisms, such as Bifidobacterium spp. and Klebsiella spp., were the predominant ones throughout the whole operation and possibly caused the unsatisfied biohydrogen production. The composition of microorganisms is the principal factor affecting biohydrogen production. Aside from the well-known hydrogen-producing Clostridium spp., several other microorganisms not only coexist but can also significantly affect system performance. The monitoring method established in the present study provides a fast quantification procedure to help operators understand how the system works and therefore quickly respond in operations.

Identification of Theileria parva and Theileria sp. (buffalo) 18S rRNA gene sequence variants in the African Buffalo (Syncerus caffer) in southern Africa

Theileria parva is the causative agent of Corridor disease in cattle in South Africa. The African buffalo (Syncerus caffer) is the reservoir host, and, as these animals are important for eco-tourism in South Africa, it is compulsory to test and certify them disease free prior to translocation. A T. parva-specific real-time polymerase chain reaction (PCR) test based on the small subunit ribosomal RNA (18S rRNA) gene is one of the tests used for the diagnosis of the parasite in buffalo and cattle in South Africa. However, because of the high similarity between the 18S rRNA gene sequences of T. parva and Theileria sp. (buffalo), the latter is also amplified by the real-time PCR primers, although it is not detected by the T. parva-specific hybridization probes. Preliminary sequencing studies have revealed a small number of sequence differences within the 18S rRNA gene in both species but the extent of this sequence variation is unknown. The aim of the current study was to sequence the 18S rRNA genes of T. parva and Theileria sp. (buffalo), and to determine whether all identified genotypes can be correctly detected by the real-time PCR assay. The reverse line blot (RLB) hybridization assay was used to identify T. parva and Theileria sp. (buffalo) positive samples from buffalo blood samples originating from the Kruger National Park, Hluhluwe-iMfolozi Park, the Greater Limpopo Transfrontier Park, and a private game ranch in the Hoedspruit area. T. parva and Theileria sp. (buffalo) were identified in 42% and 28%, respectively, of 252 samples, mainly as mixed infections. The full-length 18S rRNA gene of selected samples was amplified, cloned and sequenced. From a total of 20 sequences obtained, 10 grouped with previously published T. parva sequences from GenBank while 10 sequences grouped with a previously published Theileria sp. (buffalo) sequence. All these formed a monophyletic group with known pathogenic Theileria species. Our phylogenetic analyses confirm the distinction between Theileria sp. (buffalo) and T. parva and indicate the existence of a single group of T. parva and two Theileria sp. (buffalo) 18S rRNA gene variants in the African buffalo. Despite the observed variation in the full-length parasite 18S rRNA gene sequences, the area in the V4 hypervariable region where the RLB and real-time PCR hybridization probes were developed was relatively conserved. The T. parva specific real-time PCR assay was able to successfully detect all T. parva variants and, although amplicons were obtained from Theileria sp. (buffalo) DNA, none of the Theileria sp. (buffalo) 18S rRNA sequence variants were detected by the T. parva-specific hybridization probes.

Non‐invasive genetic identification of small mammal species using real‐time polymerase chain reaction

DNA identification of non-invasive samples is a potentially useful tool for monitoring small mammal species. Here we describe a novel method for identifying five small mammal species: wood mouse, bank vole, common shrew, pygmy shrew and water shrew. Species-specific real-time polymerase chain reaction primers were designed to amplify fragments of the mitochondrial cytochrome b gene from hair and scat samples. We also amplified nuclear DNA from scats, demonstrating their potential as a source of DNA for population genetic studies.

Development and Optimization of a Real-Time Detection Assay for Xanthomonas fragariae in Strawberry Crown Tissue with Receiver Operating Characteristic Curve Analysis

Angular leaf spot of strawberry is caused by the bacterium Xanthomonas fragariae. The disease is transmitted primarily through systemically infected nursery stock. This creates problems for nurseries wishing to export plants to Europe because of quarantine restrictions. Currently, field inspections for symptoms are used to certify plants free of X. fragariae, but visual inspection is not useful for detecting plants infected systemically. To detect systemic infections, polymerase chain reaction (PCR) is the desired tool because of its sensitivity, specificity, and ease of use. In this study, we developed three sets of real-time PCR primers and probes and determined optimal reaction conditions for use of these primers for the detection of the bacterium X. fragariae in strawberry crown tissue. Real time detection proved to be both more sensitive and specific than standard PCR. Moreover, the detection of X. fragariae in crown tissue extract was possible with real-time PCR but not with standard PCR which is a significant improvement over standard PCR. The information on sensitivity and specificity of the primer sets was used to evaluate the performance of these primers with receiver operating characteristic (ROC) curve analysis under different tolerances. The results of this analysis can be used to provide guidance on threshold selection to manage disease below unacceptable levels. The results of this research may be useful to regulators and inspectors who must certify that plants meet European and Mediterranean Plant Protection Organization standards.

Differentiation of European and North American genotypes of Phytophthora ramorum by real-time polymerase chain reaction primer extension

Phytophthora ramorum, the organism responsible for sudden oak death, causes severe damage to oak (Quercus spp.) and tanoak (Lithocarpus densiflorus) trees in California and Oregon. There are now >100 host plants that can be affected by this pathogen in nurseries and in the wild in Europe and North America. Molecular diagnosis and genotyping are important tools to monitor and prevent spread of this pathogen, to enforce quarantines, and to support eradication programs. By amplifying and sequencing DNA in a worldwide panel of strains of P. ramorum, we discovered single-nucleotide polymorphisms (SNP) in two genes (β -tubulin and cellulose binding elicitor lectin (CBEL)) that differ in the European and the North American P. ramorum isolates. Using these SNPs in diagnostic assays and distinguishing among strains of different geographic origins, we developed primer extension assays in real-time polymerase chain reaction using allele-specific oligonucleotides. All European isolates genotyped were homozygous for the SNP at position 279 in the β -tubulin gene (β- tub279 ; G/G) but heterozygous at position 858 (β- tub858 ; A/G). By contrast, all the California isolates were heterozygous for β -tub279 (T/G) but homozygous for β -tub858 (A/A). For CBEL, all European isolates were heterozygous for the SNPs at position 245 (CBEL245 ; G/C) and 412 (CBEL412 ; G/A), but the California samples were homozygous at both SNP loci CBEL245 (G/G) and CBEL412 (G/G). This assay was also used directly on DNA extracted from the ELISA lysates from field samples collected in British Columbia in 2005 by the Canadian Food Inspection Agency. Although most samples had the North American DNA profiles, there were several samples with the European profiles from the nurseries. This finding could provide information on the biology, epidemiology, etiology, host range, and populations of P. ramorum from North America and Europe and provide useful tools in P. ramorum surveys. Key words: primer extension, β- tubulin, CBEL, genotyping, allele specific oligonucleotide, SybrGreen, mating type, PCR-ASO. Le Phytophthora ramorum, l'organisme responsable de l'encre des chênes rouges ou aussi appelé la mort subite du chêne, cause de sérieux dommages aux chênes (Quercus spp.) et lithocarpes (Lithocarpus densiflorus) de la Californie et de l'Oregon. Il y a maintenant plus de 100 espèces de plantes hôtes pouvant être infectées par cet agent pathogène en Europe et en Amérique du nord, autant en pépinière qu'en nature. Le diagnostic moléculaire et le génotypage sont des outils permettant de répertorier et de prévenir la propagation de cet agent pathogène, de renforcer les mesures de quarantaines et de supporter les programmes d'éradication de la maladie. En amplifiant et en séquençant l'ADN d'une collection mondiale du P. ramorum, nous avons découvert des polymorphismes dans deux gènes (β -tubuline et CBEL « cellulose binding elicitor lectin ») pouvant différencier les isolats européens des nord- américains du P. ramorum. En utilisant ces SNP dans des tests diagnostics et en différenciant les souches de diverses origines géographiques, nous avons utilisé la méthode d'extension des amorces avec la réaction de polymerisation en chaîne (PCR) en temps-réel et des oligonucleotides spécifiques d'allèle. Tous les isolats européens génotypés étaient homozygotes pour la β- tubuline pour le SNP β -tub279 (G/G), mais hétérozygotes pour β -tub858 (A/G). Par ailleurs, tous les isolats californiens étaient hétérozygotes pour β -tub279 (T/G), mais homozygotes pour β -tub858 (A/A). Pour ce qui est de CBEL, tous les isolats européens étaient hétérozygotes pour CBEL245 (G/C) et CBEL412 (G/A), mais les échantillons de Californie étaient homozygotes aux deux SNP loci, CBEL245 (G/G) et CBEL412 (G/G). Ce test a aussi été utilisé directement sur des échantillons environnementaux d'ADN extraits de lysats d'ELISA provenant de pépinières de Colombie-Britannique dans une enquête de l'Agence canadienne d'inspection des aliments réaliseé en 2005. Presque tous ces échantillons présentaient un profile nord américain contre quelques-uns présentant un profil européen. Cette découverte est importante car elle fournit de l'information sur la biologie, l'épidémiologie, l'étiologie, la diversité d'hôtes et les populations du P. ramorum d'Europe et d'Amérique du nord. Mots-clés : extension des amorces, β- tubuline, CBEL, génotypage, oligonucléotide spécifique d'allèle, SybrGreen, type sexuel, PCR-ASO.

Enhanced Expression of Keratinocyte Growth Factor and Its Receptor Correlates with Venous Invasion in Pancreatic Cancer

Keratinocyte growth factor (KGF) and KGF receptor (KGFR) have been implicated in cancer growth as well as tissue development and repair. In this study, we examined whether KGF and KGFR have a role in human pancreatic ductal adenocarcinoma (PDAC). KGFR mRNA was expressed in eight pancreatic cancer cell lines, whereas the KGF mRNA was detected in seven of the cell lines and was absent in MIA PaCa-2 cells. KGFR and KGF immunoreactivity were localized in the cancer cells in 41.5 and 34.0% of patients, respectively. There was a significant correlation between KGFR or KGF immunoreactivity and venous invasion and a significant correlation between the presence of both markers and venous invasion, vascular endothelial growth factor (VEGF)-A expression, and poor prognosis. Exogenous KGF increased VEGF-A expression and release in MIA PaCa-2 cells, and PANC-1 cells stably transfected to overexpress KGF-exhibited increased VEGF-A expression. Moreover, short hairpin-KGFR transfection in MIA PaCa-2 cells reduced the stimulatory effect of exogenous KGF on VEGF-A expression. Short hairpin-KGF transfection in KLM-1 cells reduced VEGF-A expression in the cells. KGFR and KGF may act to promote venous invasion and tumor angiogenesis in PDAC, raising the possibility that they may serve as novel therapeutic targets in anti-angiogenic strategies in PDAC.

Multiplexed Detection of Anthrax-Related Toxin Genes

Simultaneous analysis of three targets in three colors on any real-time polymerase chain reaction (PCR) instrument would increase the flexibility of real-time PCR. For the detection of Bacillus strains that can cause inhalation anthrax-related illness, this ability would be valuable because two plasmids confer virulence, and internal positive controls are needed to monitor the testing in cases lacking target-specific signals. Using a real-time PCR platform called MultiCode-RTx, multiple assays were developed that specifically monitor the presence of Bacillus anthracis-specific virulence plasmid-associated genes. In particular for use on LightCycler-1, two triplex RTx systems demonstrated high sensitivity with limits of detection nearing single-copy levels for both plasmids. Specificity was established using a combination of Ct values and correct amplicon melting temperatures. All reactions were further verified by detection of an internal positive control. For these two triplex RTx assays, the analytical detection limit was one to nine plasmid copy equivalents, 100% analytical specificity with a 95% confidence interval (CI) of 9%, and 100% analytical sensitivity with a CI of 2%. Although further testing using clinical or environmental samples will be required to assess diagnostic sensitivity and specificity, the RTx platform achieves similar results to those of probe-based real-time systems.

Sixteen hours of fasting differentially affects hepatic and muscle insulin sensitivity in mice

Fasting readily induces hepatic steatosis. Hepatic steatosis is associated with hepatic insulin resistance. The purpose of the present study was to document the effects of 16 h of fasting in wild-type mice on insulin sensitivity in liver and skeletal muscle in relation to 1) tissue accumulation of triglycerides (TGs) and 2) changes in mRNA expression of metabolically relevant genes. Sixteen hours of fasting did not show an effect on hepatic insulin sensitivity in terms of glucose production in the presence of increased hepatic TG content. In muscle, however, fasting resulted in increased insulin sensitivity, with increased muscle glucose uptake without changes in muscle TG content. In liver, fasting resulted in increased mRNA expression of genes promoting gluconeogenesis and TG synthesis but in decreased mRNA expression of genes involved in glycogenolysis and fatty acid synthesis. In muscle, increased mRNA expression of genes promoting glucose uptake, as well as lipogenesis and beta-oxidation, was found. In conclusion, 16 h of fasting does not induce hepatic insulin resistance, although it causes liver steatosis, whereas muscle insulin sensitivity increases without changes in muscle TG content. Therefore, fasting induces differential changes in tissue-specific insulin sensitivity, and liver and muscle TG contents are unlikely to be involved in these changes.

A real-time BIO-PCR assay for detection of Ralstonia solanacearum race 3, biovar 2, in asymptomatic potato tubers

Ralstonia solanacearum, the causal agent of brown rot of potato, can often be carried latently in seed potato tubers. We designed real-time PCR (polymerase chain reaction) primers and probe and a highly sensitive BIO-PCR assay for specific detection of the strains of race 3, biovar (bv.) 2 of R. solanacearum in asymptomatic potato tubers. The biovar-specific primers and probe reacted with all 17 strains of bv. 2 of R. solanacearum tested, including 12 from potato and 5 from geranium. None of the other 35 strains of R. solanacearum reacted, including 4 strains of bv. 1 from potato or the closely related blood disease bacterium from banana. None of 13 other bacteria reacted, including 5 Erwinia and 2 Clavibacter species from potato. Using undiluted potato tuber extract, inoculated with R. solanacearum bv. 2, as few as 30 cells/mL of extract could be detected. Two of 14 naturally infected potato tubers with no disease symptoms were positive by the newly described real-time BIO-PCR assay, whereas none were positive with a standard real-time PCR assay. This is the first report of the detection of R. solanacearum bv. 2 in asymptomatic, latently infected potato tubers by PCR. The real-time assay is very simple and much less time consuming than classical PCR.

RTPrimerDB: the real-time PCR primer and probe database.

The real-time polymerase chain reaction (PCR) methodology has become increasingly popular for nucleic acids detection and/or quantification. As primer/probe design and experimental evaluation is time-consuming, we developed a public database application for the storage and retrieval of validated real-time PCR primer and probe sequence records. The integrity and accuracy of the data are maintained by linking to and querying other reference databases. RTPrimerDB provides free public access through the Web to perform queries and submit user based information. Primer/probe records can be searched for by official gene symbol, nucleotide sequence, type of application, detection chemistry, LocusLink or Single Nucleotide Polymorphism (SNP) identifier, and submitter's name. Each record is directly linked to LocusLink, dbSNP and/or PubMed to retrieve additional information on the gene/SNP for which the primers/probes are designed. Currently, the database contains primer/probe records for human, mouse, rat, fruit fly and zebrafish, and all current detection chemistries such as intercalating dyes (SYBR Green I), hydrolysis probes (Taqman), adjacent hybridizations probes and molecular beacons. Real-time PCR primer/probe records are available at http://www.realtimeprimerdatabase.ht.st.