Sensitive Quantitative Real-time PCR Assay Research Articles

Establishment of a murine model of congenital toxoplasmosis and validation of a qPCR assay to assess the parasite load in maternal and fetal tissues.

Toxoplasma gondii is the causative agent of toxoplasmosis, a disease that affects warm-blooded animals and one third of the human population worldwide. Pregnant women who have never been exposed to the parasite constitute an important risk group, as infection during pregnancy often leads to congenital toxoplasmosis, the most severe form of the disease. Current therapy for toxoplasmosis is the same as it was 50 years ago and has little or no effect when vertical transmission occurs. Therefore, it is urgent to develop new strategies to prevent mother-to-fetus transmission. The implementation of experimental animal models of congenital toxoplasmosis that reproduces the transmission rates and clinical signs in humans opens an avenue of possibilities to interfere in the progression of the disease. In addition, knowing the parasite load in maternal and fetal tissues after infection, which may be related to organ abnormalities and disease outcome, is another important step in designing a promising intervention strategy. Therefore, we implemented here a murine model of congenital toxoplasmosis with outbred Swiss Webster mice infected intravenously with tachyzoites of the ME49 strain of T. gondii that mimics the frequency of transmission of the parasite, as well as important clinical signs of human congenital toxoplasmosis, such as macrocephaly, in addition to providing a highly sensitive quantitative real-time PCR assay to assess parasite load in mouse tissues. As the disease is not restricted to humans, also affecting several domestic animals, including companion animals and livestock, they can also benefit from the model presented in this study.

Novel multiplex RT-PCR assay to detect BCR/ABL mRNA variants

The detection of the t(9;22) translocation, which results in the formation of the BCR/ABL oncoprotein, in patients diagnosed with chronic and acute leukaemias, allows for the administration of highly effective tyrosine kinase inhibitors such as imatinib and nilotinib. For the effective management of these patients it is important to monitor for minimal residual disease, allowing early therapy intervention prior to haematological relapse. Currently this is achieved through very sensitive quantitative real-time PCR assays. Unfortunately, these assays are highly specific to the BCR/ABL variant expressed: p210, p190 or p230 and require identification of the transcript type prior to selection of the correct monitoring assay. We have developed a novel multiplex BCR/ABL variant PCR assay that can identify the variant in a single qualitative assay, creating both a cost and time effective way to identify the most appropriate monitoring methodology for each patient.

Campylobacter hepaticus, the cause of spotty liver disease in chickens, is present throughout the small intestine and caeca of infected birds

Spotty liver disease (SLD) causes significant egg production losses and mortality in chickens and is therefore a disease of concern for some sectors of the poultry industry. Although the first reports of the disease came from the United States in the 1950s it is only recently that the organism that causes the disease was identified, isolated, and characterised as a new bacterial species, Campylobacter hepaticus. The first isolations of C. hepaticus were from the livers and bile of SLD affected birds. Isolates could only be recovered from samples that had a monoculture of C. hepaticus in the tissues, as a selective culturing method has not yet been developed. In non-selective growth conditions the slow growing C. hepaticus is quickly outgrown by many other members of the chicken microbiota. Therefore, it is currently not possible to use a culturing approach to evaluate C. hepaticus carriage in tissues, such as the gastrointestinal tract (GIT), that also carry complex microbial populations. As it is suspected that birds become infected via the faecal-oral route it is important that pathogen carriage in the GIT is investigated. In the present study, a specific and sensitive quantitative real-time PCR assay, based on the glycerol kinase gene of C. hepaticus, was developed. The assay facilitated the detection and quantification of C. hepaticus in tissue samples from clinical cases of SLD. It was shown that in infected birds C. hepaticus colonises the small intestine, increasing in abundance from duodenum to ileum, and is at highest levels within the ceaca. C. hepaticus was also readily detected in cloacal swabs, indicating that thecl-oral infection.

Sensitive quantification of Clostridium perfringens in human feces by quantitative real-time PCR targeting alpha-toxin and enterotoxin genes.

BackgroundClostridium perfringens is a widespread pathogen, but the precise quantification of this subdominant gut microbe remains difficult due to its low fecal count (particularly in asymptomatic subjects) and also due to the presence of abundant polymerase-inhibitory substances in human feces. Also, information on the intestinal carriage of toxigenic C. perfringens strains in healthy subjects is sparse. Therefore, we developed a sensitive quantitative real-time PCR assays for quantification of C. perfringens in human feces by targeting its α-toxin and enterotoxin genes. To validate the assays, we finally observed the occurrence of α-toxigenic and enterotoxigenic C. perfringens in the fecal microbiota of healthy Japanese infants and young adults.MethodsThe plc-specific qPCR assay was newly validated, while primers for 16S rRNA and cpe genes were retrieved from literature. The assays were validated for specificity and sensitivity in pre-inoculated fecal samples, and were finally applied to quantify C. perfringens in stool samples from apparently healthy infants (n 124) and young adults (n 221).ResultsThe qPCR assays were highly specific and sensitive, with a minimum detection limit of 103 bacterial cells/g feces. Alpha-toxigenic C. perfringens was detected in 36 % infants and 33 % adults, with counts ranging widely (103-107 bacterial cells/g). Intriguingly, the mean count of α-toxigenic C. perfringens was significantly higher in infants (6.0 ± 1.5 log10 bacterial cells/g), as compared to that in adults (4.8 ± 1.2). Moreover, the prevalence of enterotoxigenic C. perfringens was also found to be significantly higher in infants, as compared to that in adults. The mean enterotoxigenic C. perfringens count was 5.9 ± 1.9 and 4.8 ± 0.8 log10 bacterial cells/g in infants and adults, respectively.ConclusionsThese data indicate that some healthy infants and young adults carry α-toxigenic and enterotoxigenic C. perfringens at significant levels, and may be predisposed to related diseases. Thus, high fecal carriage of toxigenic C. perfringens in healthy children warrants further investigation on its potential sources and clinical significance in these subjects. In summary, we present a novel qPCR assay for sensitive and accurate quantification of α-toxigenic and enterotoxigenic C. perfringens in human feces, which should facilitate prospective studies of the gut microbiota.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0561-y) contains supplementary material, which is available to authorized users.

Airborne inoculum of Fusarium oxysporum f. sp. cucumerinum

Fusarium oxysporum f. sp. cucumerinum is the fungal pathogen responsible for Fusarium vascular wilt of cucumber. In Australia, disease management in soilless greenhouse cucumbers is limited by the understanding of the disease cycle, in particular the risk associated with infection of pruning wounds by airborne propagules. Aerial dissemination of the pathogen in response to temperature and relative humidity was investigated using a specific and sensitive quantitative real-time PCR assay. Both macroconidia and microconidia were identified as airborne propagules and a potential relationship between fluctuation in relative humidity and spore release was found, however, it appears that crop disturbance may also influence conidia liberation. Experimental inoculation of stem wounds with conidia however, failed to establish infections. These results suggest that aerial inoculum propagates and disseminates the pathogen, however airborne spores are deposited on the substrate surface and infection occurs primarily through the root. We conclude that while airborne conidia are a risk to infection of cucumber plants in soilless greenhouse crops, resistant rootstocks are likely to provide good protection against this pathogen.

Abstract B31: Establishing the use of genomic profiling on rare prostate cancer disseminated tumor cells

Abstract Objective: Dissemination of prostate cancer tumor cells remains a critical challenge in understanding disease progression and effective long-term treatment of patients. Disseminated tumor cells (DTCs) from bone marrow are resistant to chemotherapy and radiation, and represent a potential long-lived source for lethal metastases. Quantification of disseminated and circulating tumor cells provides promise as a novel diagnostic in measuring tumor burden and assessing the risk of relapse. However, very limited knowledge exists about the biology of these rare cells, including the genomic aberrations associated with clinical outcome. Methods: DTCs are immunomagnetically enriched from bone marrow aspirates, then individually captured under the microscope based on immunofluorescent staining of EpCaM cell surface molecules. We have established use of whole genome amplification and high-density SNP-CGH arrays to genomically profile rare DTC populations (n=2-25 cells). Using Fused Lasso and Nexus Copy Number software, we quantify and characterize copy number alterations and loss-of-heterozygosity regions among samples isolated from 58 patients taken at the time of radical prostectomy and from 13 patients with advanced, metastatic or lethal prostate cancer. We classify DTC aberrations by tumor stage and compare to those aberrations identified in primary and metastatic tumors. Regions of interest are validated using a highly sensitive quantitative real-time PCR assay. In addition, we have completed extensive optimization of analysis parameters to handle the increased probe variation associated with low input DNA quantity and whole genome amplification. Results: DTCs isolated at the time of radical prostatectomy from early stage prostate cancer patients are generally heterogeneous and are marked with limited focal aberrations. Focal deletions of less than 100 kb are frequent events in these samples, as are focal gains of less than 500 kb, albeit less frequently. While many of these focal copy number changes are identified in regions of common copy number variation (CNV) within the population, a considerable number are rare and unique to DTCs. Cancer-specific alterations are observed in members of the cadherin family and transcription factors SIX3, SOX4, SOX17 and GATA6. Because most abberations are patient-specific, bioinformatic analysis is used to illuminate commonly altered biological pathways. In contrast, DTCs from patients with advanced stages of prostate cancer typically show frequent and large (>1 Mb) clonal amplifications and deletions, suggesting a high degree of genomic instability that is common among these patients. Aberrant regions that are uniquely identified in DTCs from patients with advanced, metastatic and castrationresistant prostate cancers include gain of 1q43–q44 and 1q31.3–q41. In addition, several regions were found to be recurrent among at least 25% of metastatic tumors and DTCs from advanced stages, including in order of prevalence: +8q, −8p, +9q, −13, −22, −16q, −7q, −6q13–q22.31, +3q24–q26.1, +11q13.2–13.4, −17p13.3–p12 and +2p16.3–p16.2. Conclusions: We have established methods and quality control measures for genome-wide profiling of rare cell populations. These methods will be functionally useful to gain a new understanding of the genomic aberrations that drive DTC dissemination, dormancy, and metastatic reactivation in prostate cancer. Citation Format: Jamie R. Schoenborn, Jing Xia, Sandy Larson, Lisha Brown, Colm Morrissey, Paul Lange, Peter S. Nelson, Robert L. Vessella, Min Fang. Establishing the use of genomic profiling on rare prostate cancer disseminated tumor cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B31.

Microbiological safety of porcine islets: comparison with source pig

Pig islet donors intended for clinical xenotransplantation for the treatment of diabetes must meet stringent conditions. Among others, viruses with the potential to cross the species barrier should be excluded from the herd: this list includes encephalomyocarditis virus (EMCV), hepatitis E virus (HEV), porcine cytomegalovirus (PCMV) and porcine γ-lymphotropic herpesvirus (PLHV). As an islet product is isolated from the pancreas and then subjected to culture before implantation, the question is raised whether islets could be negative even if the animal itself is positive for a distinct pathogen. To answer this question, sensitive quantitative real-time PCR assays were established for EMCV, HEV, PCMV and PLHV. Twelve adult animals from a high-hygienic herd were then evaluated; testing tissues, where the virus is expected to reside in latent infection, testing islets immediately after isolation, and then isolated islets after a 7-day culture. None of the tissues tested positive for EMCV, HEV or PLHV. PCMV was observed in spleen tissue from six animals: three of these six animals were positive for isolated islets, and two of these three cases were also positive for islets after culture. Older animals in particular showed positivity, and within a given litter not all animals were PCMV positive. These data fit with spread through the herd by horizontal transmission, not in utero infection. PCMV has to be excluded from the herd to ensure that islets for transplantation are negative for PCMV.

Carrier frequency of SMA by quantitative analysis of the SMN1 deletion in the Iranian population

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder. Carrier frequency studies of SMA have been reported for various populations. Although no large-scale population-based studies of SMA have been performed in Iran, previous estimates have indicated that the incidence of autosomal recessive disorder partly because of the high prevalence of consanguineous marriage is much higher in the Iranian population than in other populations. In this study, we used a reliable and highly sensitive quantitative real-time PCR assay with SYBR green I dye to detect the copy number of the SMN1 gene to determine the carrier frequency of SMA in 200 healthy unrelated, non-consanguineous couples from different part of Iran. To validate the method in our samples, we determined the relative quantification (RQ) of patients with homozygous deletion (0.00) and hemyzygous carriers (0.29-0.55). The RQ in 10 of 200 normal individuals were within the carrier range of 0.31-0.57, estimating a carrier frequency of 5% in the Iranian population. Our data show that the SMA carrier frequency in Iran is higher than in the European population and that further programs of population carrier detection and prenatal testing should be implemented.

A highly sensitive quantitative real-time PCR assay based on the groEL gene of contemporary Thai strains of Orientia tsutsugamushi

Partial nucleotide sequences (459 bp) of the groEL gene (encoding the 60-kDa heat shock protein, HSP60) from 23 contemporary isolates of Orientiatsutsugamushi isolated from patients with acute scrub typhus in Thailand were compared with 16 reference strain sequences to evaluate the potential of groEL as a conserved and representative target for molecular diagnostics‥ Overall nucleotide identity within all available O. tsutsugamushi isolates (n = 39) was 98.8% (range: 95.0–100), reflecting a high degree of conservation; nucleotide identities were 67.5% and 65.6%, respectively, when typhus and spotted fever group rickettsiae were included‥ A highly sensitive and quantitative real-time PCR assay was designed and evaluated using 61 samples, including buffy coats from patients in Thailand and Laos. Reliable and accurate quantitation of bacterial loads allows further investigation of other diagnostic methods and may lead to an improved understanding of the pathophysiology of acute scrub typhus, an important but under-recognized disease.

Vaccination and Timing Influence SIV Immune Escape Viral Dynamics In Vivo

CD8+ cytotoxic T lymphocytes (CTL) can be effective at controlling HIV-1 in humans and SIV in macaques, but their utility is partly offset by mutational escape. The kinetics of CTL escape and reversion of escape mutant viruses upon transmission to MHC-mismatched hosts can help us understand CTL-mediated viral control and the fitness cost extracted by immune escape mutation. Traditional methods for following CTL escape and reversion are, however, insensitive to minor viral quasispecies. We developed sensitive quantitative real-time PCR assays to track the viral load of SIV Gag164–172 KP9 wild-type (WT) and escape mutant (EM) variants in pigtail macaques. Rapid outgrowth of EM virus occurs during the first few weeks of infection. However, the rate of escape plateaued soon after, revealing a prolonged persistence of WT viremia not detectable by standard cloning and sequencing methods. The rate of escape of KP9 correlated with levels of vaccine-primed KP9-specific CD8+ T cells present at that time. Similarly, when non-KP9 responder (lacking the restricting Mane-A*10 allele) macaques were infected with SHIVmn229 stock containing a mixture of EM and WT virus, rapid reversion to WT was observed over the first 2 weeks following infection. However, the rate of reversion to WT slowed dramatically over the first month of infection. The serial quantitation of escape mutant viruses evolving during SIV infection shows that rapid dynamics of immune escape and reversion can be observed in early infection, particularly when CD8 T cells are primed by vaccination. However, these early rapid rates of escape and reversion are transient and followed by a significant slowing in these rates later during infection, highlighting that the rate of escape is significantly influenced by the timing of its occurrence.

Assembly of hepatitis delta virus: particle characterization, including the ability to infect primary human hepatocytes.

Efficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins of hepatitis B virus (HBV), the natural helper of HDV, and another to initiate replication of the HDV RNA genome. HDV released into the media was assayed for HDV RNA and HBV envelope proteins and characterized by rate-zonal sedimentation, immunoaffinity purification, electron microscopy, and the ability to infect primary human hepatocytes. Among the novel findings were that (i) immunostaining for delta antigen 6 days after infection with 300 genome equivalents (GE) per cell showed only 1% of cells as infected, but this was increased to 16% when 5% polyethylene glycol was present during infection; (ii) uninfected cells did not differ from infected cells in terms of albumin accumulation or the presence of E-cadherin at cell junctions; and (iii) sensitive quantitative real-time PCR assays detected HDV replication even when the multiplicity of infection was 0.2 GE/cell. In the future, this HDV assembly and infection system can be further developed to better understand the mechanisms shared by HBV and HDV for attachment and entry into host cells.