Use of real-time quantitative PCR to detect Chlamydophila felis infection.
Ontology highlight
ABSTRACT:
SUBMITTER: Helps C
PROVIDER: S-EPMC88209 | biostudies-literature | 2001 Jul
REPOSITORIES: biostudies-literature
Publications
Use of real-time quantitative PCR to detect Chlamydophila felis infection.
Helps C C Reeves N N Tasker S S Harbour D D
Journal of clinical microbiology 20010701 7
A real-time PCR assay was developed to detect and quantify Chlamydophila felis infection of cats. The assay uses a molecular beacon to specifically identify the major outer membrane protein gene, is highly reproducible, and is able to detect fewer than 10 genomic copies. ...[more]
Similar Datasets
Project description:Fifteen cats infected with Chlamydophila felis were monitored for the presence of C. felis DNA on ocular swabs by using real-time PCR and for clinical signs of disease. The cats were assigned to three groups: oral doxycycline at 10 mg/kg of body weight/day for 7 days (six cats), oral doxycycline at 10 mg/kg/day for 14 days (five cats), and an untreated control group (four cats). The untreated cats remained positive for C. felis throughout the trial; clinical signs were most severe on days 14 to 21 postinfection, and then they declined. Treatment with 7 and 14 days of doxycycline decreased C. felis relative copy numbers and clinical signs rapidly. C. felis became undetectable in some of the cats during or after treatment. However, after the cessation of treatment, a recurrence of high relative copy numbers of C. felis and severe clinical signs in all cats was seen. Rescue treatment with 21 days of doxycycline was successful at eliminating infection in eight of the cats; a further 28 days of doxycycline was required to eliminate infection in the remaining three cats. It was concluded that 7, 14, and, in some cases, 21 days of treatment with oral doxycycline will not eliminate C. felis infection. At least 28 days of treatment with doxycycline is required to ensure elimination of the organism. Real-time PCR is a sensitive technique for monitoring C. felis infection and the response to antibiotic treatment.
| S-EPMC1081369 | biostudies-literature
Project description:Chlamydophila felis and feline herpesvirus (FHV) are pathogens commonly associated with feline respiratory and ocular disease. A real-time multiplex PCR assay was developed to allow detection of these organisms, together with feline 28S ribosomal DNA, in a single tube. Of 538 ocular swab samples tested, 123 were positive for FHV, 97 were positive for C. felis, and 16 were positive for both pathogens.
| S-EPMC156487 | biostudies-literature
Project description:We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/ microl to 1 ng/ microl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/ microl to 1 ng/ microl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/ microl to 1 ng/ microl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/ microl to 1 ng/ microl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler and the LightCycler platforms. The assay may be useful for the early detection of smallpox virus infections should such infections occur as a result of a deliberate or an accidental recurrence.
| S-EPMC179856 | biostudies-literature
Project description:22q11.2 Deletion syndrome (22q11.2 DS) [DiGeorge syndrome type 1 (DGS1)] occurs in ?1:3,000 live births; 75% of children with DGS1 have severe congenital heart disease requiring early intervention. The gold standard for detection of DGS1 is fluorescence in situ hybridization (FISH) with a probe at the TUPLE1 gene. However, FISH is costly and is typically ordered in conjunction with a karyotype analysis that takes several days. Therefore, FISH is underutilized and the diagnosis of 22q11.2 DS is frequently delayed, often resulting in profound clinical consequences. Our goal was to determine whether multiplexed, quantitative real-time PCR (MQPCR) could be used to detect the haploinsufficiency characteristic of 22q11.2 DS. A retrospective blinded study was performed on 382 subjects who had undergone congenital heart surgery. MQPCR was performed with a probe localized to the TBX1 gene on human chromosome 22, a gene typically deleted in 22q11.2 DS. Cycle threshold (C(t)) was used to calculate the relative gene copy number (rGCN). Confirmation analysis was performed with the Affymetrix 6.0 Genome-Wide SNP Array. With MQPCR, 361 subjects were identified as nondeleted with an rGCN near 1.0 and 21 subjects were identified as deleted with an rGCN near 0.5, indicative of a hemizygous deletion. The sensitivity (21/21) and specificity (361/361) of MQPCR to detect 22q11.2 deletions was 100% at an rGCN value drawn at 0.7. One of 21 subjects with a prior clinical (not genetically confirmed) DGS1 diagnosis was found not to carry the deletion, while another subject, not previously identified as DGS1, was detected as deleted and subsequently confirmed via microarray. The MQPCR assay is a rapid, inexpensive, sensitive, and specific assay that can be used to screen for 22q11.2 deletion syndrome. The assay is readily adaptable to high throughput.
| S-EPMC2957771 | biostudies-literature
Project description:Human infection with Chlamydophila (Chlamydia) psittaci can lead to psittacosis, a disease that occasionally results in severe pneumonia and other medical complications. C. psittaci is currently grouped into seven avian genotypes: A through F and E/B. Serological testing, outer membrane protein A (ompA) gene sequencing, and restriction fragment length polymorphism analysis are currently used for distinguishing these genotypes. Although accurate, these methods are time-consuming and require multiple confirmatory tests. By targeting the ompA gene, a real-time PCR assay has been developed to rapidly detect and genotype C. psittaci by light-upon-extension chemistry and high-resolution melt analysis. Using this assay, we screened 169 animal specimens; 98 were positive for C. psittaci (71.4% genotype A, 3.1% genotype B, 4.1% genotype E, and 21.4% unable to be typed). This test may provide insight into the distribution of each genotype among specific hosts and provide epidemiological and epizootiological data in human and mammalian/avian cases. This diagnostic assay may also have veterinary applications during chlamydial outbreaks, particularly with respect to identifying the sources and tracking the movements of a particular genotype when multiple animal facilities are affected.
| S-EPMC2620869 | biostudies-literature
Project description:A real-time PCR was designed for detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae such that each pathogen could be detected in a single tube and differentiated using molecular beacons marked with different fluorochromes. This duplex PCR, targeting the P1 adhesion gene for M. pneumoniae and the ompA gene for C. pneumoniae, was compared with two conventional PCR assays targeting the 16S rRNA gene and the ompA gene. A total of 120 clinical throat and nasopharyngeal swab samples were tested. DNA extraction was performed using an alkali denaturation/neutralization method, and real-time amplification, detection, and data analysis were performed using a Rotor-Gene 2000 real-time rotary analyzer (Corbett Life Science, Sydney, Australia). Using conventional PCR as a reference in an analysis of 120 samples, 13 of 14 samples positive for C. pneumoniae were detected by the novel real-time PCR. In an analysis of M. pneumoniae, 22 samples were positive in the conventional PCR and the novel assay detected 24 positive samples. When using the conventional PCR as a reference, sensitivity and specificity were 93% and 100%, respectively, for C. pneumoniae and 100% and 98%, respectively, for M. pneumoniae. With an overall agreement of 98.8%, this suggests that performance of the new duplex real-time PCR is comparable to that of conventional PCR.
| S-EPMC2238133 | biostudies-literature
Project description:Age-matched BALB/c female mice were implanted subcutaneously with slow release estradiol (E2) pellets (35 mg/pellet/mouse) or similar-sized placebo pellets .One week post-implantation, mice were infected intranasally with a sublethal dose of H5N1 A/Vietnam/1203/2004 vaccine reassortant bearing a monobasic HA cleavage site. Mouse lungs were harvested on day 1 post infection and total RNA was extracted using QIAgen RNeasy microarray tissue mini kit. A total of 100 µg high-quality RNA was reverse transcribed using RT2 Easy First Strand Kit (QIAgen). Resultant cDNA was used as the template along with Profiler™ PCR Array Mouse Signal Transduction PathwayFinder™ kit (QIAgen) to quantitate gene expression activated by E2 in response to H5N1 infection.
2019-09-11 | GSE128447 | GEO
Project description:The mink circovirus (MiCV), a newly discovered pathogen, is associated with diarrhea in farmed minks. The prevalence and economic importance of this virus remain poorly understood, and a quantitative method for diagnosis of MiCV infection has not been established. This research aims to develop a highly specific, sensitive, and quantitative assay for MiCV. A Real-Time quantitative polymerase chain reaction (qPCR) assay was developed to detect different isolates of the MiCV in mink samples. The qPCR system is highly sensitive with a detection limit of as low as 10 viral DNA copies. The specificity of this qPCR assay was supported by the absence of cross-reaction with other pathogens. The coefficients of variation were low for both inter-assay and intra-assay variabilities. In addition, the results also expressed the distribution of MiCV in infectious mink tissues with high levels of virus in the skeletal muscle and heart. The heart occupied a higher proportion than other tissues, which can be considered the primary source of test material. This qPCR method could be a useful tool for epidemiological studies and disease management. This method for MiCV is highly specific, sensitive, repeatable, quantitative, and can rapidly determine viral load levels in different tissues samples.
| S-EPMC5960700 | biostudies-literature
Project description:Pediatric astrocytomas, a leading cause of death associated with cancer, are the most common primary central nervous system tumors found in children. Most studies of these tumors focus on adults, not children. We examined the global protein and microRNAs expression pattern by 2D SDS-PAGE, mass spectrometry (MALDI-TOF) and RT2 miRNA PCR Array System. MicroRNAs analysis revealed for the first time novel microRNAs involved in astrocytomas biology. Interestingly, miR-138 and miR-145 down-regulation appear to be associated with protein over-expression of vimentin and Bax, respectively. In conclusion, our results show that novel proteins and microRNAs altered on pediatric astrocytoma could serve as biomarkers to distinguish between astrocytoma grades. Astrocytoma samples were colected from patients and total RNA isolation (30 mg of tissue) was performed using the TRIzol® protocol (Invitrogen, USA) according to the manufacturer′s instructions. Samples were analyzed using SA Biosciences RT2 miRNA PCRArray System to determied the miRNA expression between control samples and tumors RT2 miRNA PCR Array. Eigth tumor samples and two control tissue (including two control tissue replicates) were used as indicated in the sumary. A total of 3ug of RNA from each tumr samples and control tissue were placed in the PCR Array
| S-ECPF-GEOD-44280 | biostudies-other
Project description:Pediatric astrocytomas, a leading cause of death associated with cancer, are the most common primary central nervous system tumors found in children. Most studies of these tumors focus on adults, not children. We examined the global protein and microRNAs expression pattern by 2D SDS-PAGE, mass spectrometry (MALDI-TOF) and RT2 miRNA PCR Array System. MicroRNAs analysis revealed for the first time novel microRNAs involved in astrocytomas biology. Interestingly, miR-138 and miR-145 down-regulation appear to be associated with protein over-expression of vimentin and Bax, respectively. In conclusion, our results show that novel proteins and microRNAs altered on pediatric astrocytoma could serve as biomarkers to distinguish between astrocytoma grades. Astrocytoma samples were colected from patients and total RNA isolation (30 mg of tissue) was performed using the TRIzolM-BM-. protocol (Invitrogen, USA) according to the manufacturerM-bM-^@M-2s instructions. Samples were analyzed using SA Biosciences RT2 miRNA PCRArray System to determied the miRNA expression between control samples and tumors RT2 miRNA PCR Array. Eigth tumor samples and two control tissue (including two control tissue replicates) were used as indicated in the sumary. A total of 3ug of RNA from each tumr samples and control tissue were placed in the PCR Array
2013-02-12 | E-GEOD-44280 | biostudies-arrayexpress