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PubMed1995

Assessment of stress gene mRNAs (HSP-27, 60 and 70) in obstructed rabbit urinary bladder using a semi-quantitative RT-PCR method.

Stress proteins (HSPs) participate in the cellular response to various stresses including hyperthermia, hypoxia and injury. A previous work using northern blot analysis demonstrated increased expression of stress protein 70 (HSP-70) in rabbit bladder tissue subjected to partial outlet obstruction. In order to determine if the increased expression was specific for HSP-70 or, alternatively, indicated a generalized stress protein response, a modified quantitative RT-PCR technique was used to quantitate HSP mRNAs (HSP-27, 60, and 70) in normal and obstructed rabbit urinary bladder tissues. The results show the following: 1) The modified semi-quantitative RT-PCR is a sensitive and reproducible technique for detecting mRNA in bladder tissue. 2) Constitutive levels of HSP-27, HSP-60, and HSP-70 mRNAs were detected in control bladder tissues; the relative signal intensity was highest for HSP-70 and lowest for HSP-27. 3) A transient increase in HSP mRNAs was observed after obstruction; the mRNAs of HSP-27, 60 and 70 increased 4.3-, 5.6-, and 2.4-fold, respectively, at 24 h following obstruction, then gradually returned to control levels by the end of one week post-obstruction and remained stable up to 14 days post-obstruction. These data indicate that the modified quantitative RT-PCR is a useful technique for detecting mRNA in bladder tissue; the stress response which occurs in rabbit urinary bladder tissue following partial outlet obstruction is a general phenomenon.

Animals↗
PubMed2005

Microsatellite instability and gene mutations in transforming growth factor-beta type II receptor are absent in small bowel carcinoid tumors.

BACKGROUND: Microsatellite instability (MSI) with concomitant mutations in the coding region of transforming growth factor-beta type II receptor (TGFbetaRII) results in an aberrant growth-regulatory phenotype in colorectal carcinomas. The authors postulated that a similar mechanism occurred during the malignant evolution of small bowel carcinoid tumors. METHODS: Mutational analysis of two coding regions in the TGFbetaRII gene associated with MSI and BAT-26 within intron 5 of the mismatch repair gene, hMSH2, was undertaken in small bowel carcinoids (n = 14), lymph node metasasis (n = 1) and liver metastases (n = 5). Quantitative PCR analysis [TAQMAN, Applied Biosystems, Foster City, CA] was then undertaken to examine gene alterations in mismatch repair genes (hMLH1 and hMSH2) in small bowel carcinoids (n = 7) and matched normal mucosa (n = 5). Staining was then analyzed using quantitative tissue array profiling (AQUA analysis) in a small bowel EC carcinoid tissue microarray (n = 55 tumors) with immunostaining against TGFbetaRII and MSH2. RESULTS: Mutational examination of the TFGbetaRII gene and BAT-26 demonstrated that MSI was not present in any carcinoid material. Q RT-PCR analysis demonstrated statistically significant increased message levels of hMSH2 but not hMLH1 in carcinoid tumors. Quantitative analysis of membrane TGFbetaRII immunostaining using AQUA demonstrated that TGFbetaRII expression was down-regulated (P < 0.0002) in thirty-three primary small bowel carcinoids that exhibited lymph node and liver metastases compared to normal mucosa. AQUA analysis of nuclear MSH2 immunostaining demonstrated no differences for MSH2 between normal tissue and carcinoid tumor metastasis. Small bowel carcinoids characterized by variable expression of TGFbetaRII, did not exhibit MSI and had no differences in MSH2 expression. CONCLUSIONS: The molecular events leading to the formation of carcinoid tumors in the small bowel were different from those resulting in epithelial carcinomas. The usually slow-growing and relatively nonaggressive carcinoid tumors had variable expression of TGFbetaRII but were associated with the retention of mismatch repair protein function and a microsatellite-stable phenotype.

Adult↗
PubMed1996

Quantitative multiple competitive PCR of HIV-1 DNA in a single reaction tube.

A quantitative multiple competitive PCR (QMC-PCR) for determination of DNA copy numbers is described. Four competitive DNA templates for the env region of HIV-1 were constructed with sizes longer (187 and 163 bp) or shorter (122 and 105 bp) than the 142 bp of the wild-type PCR product. Varying amounts of each of these competitors are introduced together with the sample into a single reaction tube. Since competitors and wild-type fragments share the same primer recognition sequence (SK68/SK69), amplification occurs according to the rate of the introduced copy numbers. The PCR products are run on an agarose gel, and the copy number of the sample is determined by analyzing the bands with a video densitometer and calculating the equivalence point in a linear regression plot.

Binding, Competitive↗
PubMed2005

Quantification of Bifidobacterium spp., Escherichia coli and Clostridium difficile in faecal samples of breast-fed and formula-fed infants by real-time PCR.

To determine the influence of either exclusive breast-feeding or formula feeding on both composition and quantity of the gut microbiota in infants, we have developed real-time, quantitative PCR assays for the detection of Bifidobacterium spp. and Clostridium difficile. Furthermore, we have monitored the prevalence and counts of Escherichia coli by applying a previously described real-time PCR assay. We found all 100 infants tested to be colonized by Bifidobacterium spp. The bifidobacterial counts were comparable between the 50 breast-fed and 50 formula-fed infants with median values of 10.56 log10 and 10.24 log10 CFU g(-1) wet weight faeces, respectively. C. difficile was detected in 14% of the breast-fed and 30% of the formula-fed infants. In addition, the C. difficile counts were significantly lower in breast-fed infants than in the formula-fed group (median values of 3.28 log10 and 7.43 log10 CFU g(-1), respectively; p=0.03). The prevalence of E. coli in the breast-fed and formula-fed group was 80% and 94%, respectively. Also, the E. coli counts in colonized infants was significantly lower in the breast-fed infants than in the formula-fed group (median values of 9.11 log10 and 9.57 log10 CFU g(-1), respectively; p=0.004). We conclude that the prevalence and counts of C. difficile as well as E. coli are significantly lower in the gut microbiota of breast-fed infants than in that of formula-fed infants, whereas the prevalence and counts of Bifidobacterium spp. is similar among both groups.

Bifidobacterium↗
PubMed2005

Experience from the development of a diagnostic single tube real-time PCR for human caliciviruses, Norovirus genogroups I and II.

Detection of caliciviruses requires high mutation tolerance and throughput. The development of a rational simple, single tube reverse transcription-real-time quantitative PCR (QPCR) technique for human noroviruses (NV) is reported here. A dual-probe, triple-primer system (NM system) was used for simultaneous detection and preliminary differentiation of NV genogroups in fecal samples. The design was based on a comprehensive analysis of all 1140 NV sequences available in GenBank. A touch-down amplification protocol improved the frequency of detection. The final QPCR was evaluated with 71 fecal samples from outbreak and sporadic cases in Sweden (1997-2004), all calicivirus-positive by electron microscopy. Up to 56 (79 %) were positive. The method is more rational than NV detection methods described previously, and should be a developmental basis for large-scale routine methods for detection of NV.

Adult↗
PubMed2000

Quantitative RT-PCR measurement of human cytochrome P-450s: application to drug induction studies.

A quantitative RT-PCR assay has been developed that is able to measure the mRNA content of the major human CYPs (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5). The technique is highly specific, reproducible, rapid, and sensitive enough to quantitate low and high abundant mRNAs. The PCR primers were selected to specifically match each CYP mRNA, to have a very close annealing temperature, and to render PCR products of similar sizes. The PCR conditions were designed to allow the simultaneous measurement of the various human liver CYPs in a single run. To achieve precise and reproducible quantitation of each cytochrome mRNA, a external standard (luciferase mRNA) is added to the probes to monitor the efficiency of the RT step. The degree of amplification is estimated using appropriate cDNA standards and quantitation of the amplified products by fluorescent measurement. This assay can be used to quantify the most relevant CYPs in human liver and cultured human hepatocytes. CYPs 3A4 and 2E1 were the most abundant mRNAs in human liver (2.5 and 1.7 x 10(8) molecules/microgram of total RNA respectively), whereas 1A1 and 2D6 were the least abundant isoforms (1.2 and 2.1 x 10(6) molecules/microgram of total RNA). A similar pattern was also found in short-term cultured human hepatocytes. This technique is also suitable for assessing CYP mRNA induction by xenobiotics. Cells exposed to 3-methylcholanthrene showed a characteristic increased expression of CYP1A2 and 1A1 mRNAs. Upon incubation with phenobarbital and rifampin (rifampicin), human hepatocytes increased CYP 2B6, 3A4, and 3A5 among others.

Actins↗
PubMed2005

Molecular cloning and characterization of the clock gene period2 in the testis of lizard Podarcis sicula and its expression during seasonal reproductive cycle.

Clock genes are known to oscillate with circadian rhythmicity in the central clock structure, the suprachiasmatic nucleus of the hypothalamus, and also in peripheral tissues. Reproduction is a peripheral activity that is strongly influenced by a circadian clock in many organisms. Most mammals that exhibit a seasonal cycle are able to decode the daily changes in light across the year and to translate these in hormonal signals that regulate reproductive cycles. Expression of many clock genes has been revealed in mouse testis, although transcription of these genes seems to be constitutive in 24 h, suggesting that these genes may play in the testis a different role with regard to the central clockwork function. The seasonal breeding lizard Podarcis sicula represents an attractive model for studying some developmental and differentiation phenomena, such as gonadal maturation, since in the adult male the testis shows a spring full activity and a complete summer regression. Experimental data seem to suggest that in lizard the environmental factors, as photoperiod and temperature, affect the endogenous elements, although the interaction mechanisms are unknown. It is known that temperature signals have a direct influence on clock processes such as transcription, translation, protein phosphorylation and degradation. In addition, most data show that the expression of circadian clock genes, such as period2, is affected by length of photoperiod. In this way, the core clockwork may also decode seasonal information. Here we report the cloning, sequencing and bioinformatic analysis of period2 gene, isolated from the testis of lizard P. sicula, and its expression both in the testis and in other tissues during the different phases of the seasonal cycle. RT-PCR assays enlighten the presence of transcript in testis, brain, heart, liver and kidney in all the phases analysed. Moreover, real time quantitative PCR assays detect a peak of per2 testicular expression during gonadal regression. Our preliminary results cannot clearly demonstrate the involvement of per2 gene in seasonal reproductive cycle of male lizard P. sicula, but its presence in the testis may suggest a role of this gene during spermatogenesis. Besides, our work can provide numerous starting points to clarify the role of per2 during seasonal reproductive cycle.

Amino Acid Sequence↗
PubMed2005

Combined megaplex TCR isolation and SMART-based real-time quantitation methods for quantitating antigen-specific T cell clones in mycobacterial infection.

Despite recent advances in measuring cellular immune responses, the quantitation of antigen-specific T cell clones in infections or diseases remains challenging. Here, we employed combined megaplex TCR isolation and SMART-based real-time quantitation methods to quantitate numerous antigen-specific T cell clones using limited amounts of specimens. The megaplex TCR isolation covered the repertoire comprised of recombinants from 24 Vbeta families and 13 Jbeta segments, and allowed us to isolate TCR VDJ clonotypic sequences from one or many PPD-specific IFNgamma-producing T cells that were purified by flow cytometry sorting. The SMART amplification technique was then validated for its capacity to proportionally enrich cellular TCR mRNA/cDNA for real-time quantitation of large numbers of T cell clones. SMART amplified cDNA was shown to maintain relative expression levels of TCR genes when compared to unamplified cDNA. While the SMART-based real-time quantitative PCR conferred a detection limit of 10(-5) to 10(-6) antigen-specific T cells, the clonotypic primers specifically amplified and quantitated the target clone TCR but discriminated other clones that differed by >or=2 bases in the DJ regions. Furthermore, the combined megaplex TCR isolation and SMART-based real-time quantiation methods allowed us to quantitate large numbers of PPD-specific IFNgamma-producing T cell clones using as few as 2 x 10(6) PBMC collected weekly after mycobacterial infection. This assay system may be useful for studies of antigen-specific T cell clones in tumors, autoimmune and infectious diseases.

Animals↗
PubMed2002

Thermal injury induces expression of CD14 in human skin.

BACKGROUND: Skin is equipped with an array of immune mediators aimed at fighting invading microbes. CD14 has been shown to play a key role in modulating the activation of cells by LPS. Since LPS levels within burn wounds are often found to be elevated, we sought to examine the expression of CD14 within human skin following thermal injury. METHODS: Patients who sustained partial thickness burns, were recruited into the study (n=57). Total RNA was isolated from both burn and normal (control) skin. Northern blot analysis and TaqMan RT-PCR were used to determine skin CD14 mRNA levels. Immunohistochemistry was used to localize CD14 expression in burned and normal skin. RESULTS: Quantitative PCR showed significantly increased CD14 expression levels in the immediate post-burn period (P<0.05 burn versus non-burn). Immunohistochemistry revealed more pronounced CD14 staining 24 h after the injury, reaching normal levels approximately 5-7 days post-burn. CONCLUSION: CD14 expression peaks within the first week post-burn before declining, reaching normal levels after 14 days. This loss of supranormal CD14 expression locally within the wound may contribute to a weakened host defense response 5-6 days after injury, when patients become especially vulnerable to infection.

Adolescent↗
PubMed1999

Autografting with CD34+ peripheral blood stem cells: retained engraftment capability and reduced tumour cell content.

The efficacy of an immunomagnetic purging method and the Isolex 300 devices were assessed for selecting CD34+ cells from leukapheresis products of 29 patients with non-Hodgkin's lymphoma (NHL), 39 with multiple myeloma and 34 with breast cancer. The mean purity of the CD34+ cell population was 93.6% and the mean recovery was 67.7%. Following enzymatic cleavage by chymopapain the expression of Thy-1 and Leu-8 was significantly reduced without affecting haematological recovery. The population of selected CD34+ cells of 4/8 patients with follicular lymphoma became PCR-negative. A 2.5 log reduction of tumour cells could be achieved in four patients with multiple myeloma as shown by a quantitative PCR assay. There were no tumour cells detectable in any of the 19 CD34+ cell preparations of patients with breast cancer. In 64 patients who received 94 cycles of high-dose therapy, a mean number of 4.7x 10(6) CD34+ cells/kg were autografted. The time needed for platelet reconstitution was different when a comparison was made with 156 patients, who had received unmanipulated leukapheresis products (10 v 12 d, P = 0.006). No significant differences with regard to neutrophil recovery were noted. Five patients had a graft failure. Two of them died (on day 78 and 88 following PBSCT), and three patients were rescued with unmanipulated back-up transplants. In conclusion, the immunomagnetic selection of CD34+ cells provides autografts with reduced tumour cell content and an engraftment ability similar to that of unmanipulated autografts.

Antigens, CD34↗
PubMed1999

Hepatitis C viral load, genotype and histological severity in patients with bleeding disorders.

We report the relationship between hepatitis C virus (HCV) titre, liver histology and HCV genotype in patients with bleeding disorders. One hundred and thirty-two RIBA-2-positive patients, including 56 who were also HIV positive, were identified at our centre. Fifty of these patients, including nine who were HIV infected, underwent percutaneous liver biopsy. Liver histology was assessed using a modified histological activity index (HAI). Qualitative serum HCV PCR was positive in 87 (87%) of the 101 patients tested including 43 of 50 biopsied patients. HCV RNA titres, measured by quantitative PCR, were significantly higher in HIV-positive patients compared with HIV-negative patients (P < 0.05) but were not related to HAI, mean factor concentrate usage, duration of HCV infection or HCV genotype. There was no relationship between HCV genotype and HAI. Qualitative HCV PCR was positive in 30 of 43 liver biopsies tested. Biopsy PCR-positive and -negative cohorts were not distinguished by HAI or serum HCV titre. We conclude that although serum HCV PCR is useful in confirming the presence of HCV infection in patients with bleeding disorders, little meaningful information concerning the severity of the disease can be obtained from serum HCV quantification.

Adult↗
PubMed2006

The bacteriology of biopsies differs between newly diagnosed, untreated, Crohn's disease and ulcerative colitis patients.

The bacterial community (microbiota) that inhabits the gut of humans appears to be an important source of antigens that drive the chronic immunological processes characteristic of Crohn's disease (CD) and ulcerative colitis (UC). Most of the members of the microbiota have not yet been cultured, but nucleic-acid-based methods of detection and enumeration can provide information about the community. This investigation used these methods to obtain information about the bacteria associated with mucosal surfaces in the gut of 20 CD and 15 UC patients. Biopsies were collected from inflamed and non-inflamed sites in the intestines of newly diagnosed, untreated patients. Biopsies were also collected from several intestinal sites of 14 healthy subjects. The bacterial collections associated with the biopsies were analysed by generating PCR/denaturing gradient gel electrophoresis (DGGE) profiles, the preparation of 16S rRNA gene clone libraries, and qualitative PCR to detect specific groups of bacteria. The total numbers of bacteria associated with the biopsies were determined by real-time quantitative PCR. DGGE profiles generated from the terminal ileum and various colonic regions were characteristic of each individual but differed between subjects. DGGE profiles and 16S rRNA gene libraries showed that the bacteria associated with inflamed and non-inflamed tissues did not differ. UC patients had more bacteria associated with biopsies than did CD patients (P<0.01). Statistical analysis of the composition of 16S rRNA gene libraries showed that the bacterial collections in UC and CD patients differed (P<0.05). Unclassified members of the phylum Bacteroidetes were more prevalent in CD than in UC patients. Therefore, the types and numbers of bacteria associated with biopsy samples were distinctly different for UC and CD patients. The observations made in this study should permit targeting of specific bacteriological abnormalities in investigations of the pathogenesis of inflammatory bowel diseases and provide targets for medical interventions.

Adolescent↗
PubMed1993

A PCR-aided transcript titration assay revealing very low expression of a gene at band 3p21 in 33 cells lines derived from all types of lung cancer.

We have developed a general PCR-based method to quantify the amount of a specific mRNA present in a given cell line or tissue. We applied this quantitative PCR to analyse the expression of D8, a human gene which we recently identified in the chromosomal region 3p21, the common deletion region of lung cancer. Our PCR-aided assay shows that in most lung-cancer-derived cell lines the amount of D8 transcripts is only 2% or less of that in normal lung tissue. The virtual absence of expression may imply some role of the gene in the development of lung cancer.

Base Sequence↗
PubMed2002

Developmental changes in expression of myeloid cell leukemia-1 in human germ cells during oogenesis and early folliculogenesis.

The regulation of germ cell number in the developing ovary is central to female reproduction. Members of the Bcl-2 family of proapoptotic and antiapoptotic proteins have been implicated in this process in rodents. We investigated the expression of Mcl-1, Bcl-2, Bax, and BAD at 13-21 gestational wk in the human fetal ovary and of Mcl-1 in the adult ovary. mRNA expression of Mcl-1 and its short form Mcl-1s, Bcl-2, Bax, and BAD was demonstrated in fetal ovary by RT-PCR. Hybridization array analysis suggested a selective increase in Mcl-1 expression between 14 and 18 wk gestation, which was confirmed by quantitative PCR. There was a corresponding change in the expression of Mcl-1 protein, detected by immunohistochemistry, from germ cells at the periphery of the ovary at 14-16 wk to the largest germ cells, including oocytes within newly formed primordial follicles, at 21 wk. Mcl-1 was also expressed by oocytes of primordial and preantral follicles in the adult. Bax and BAD immunostaining was detected in both somatic and germ cells in the fetal ovary, whereas Bcl-2 was restricted to somatic cells: no changes in expression were observed. Apoptotic cells, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, were observed in all fetal ovaries but were infrequent. These results confirm that Bcl-2 family members are differentially expressed in several cell types within the developing human ovary. Increased mRNA expression and the changing distribution of Mcl-1 in germ cells as they develop into primordial follicles as well as persistence in the growing oocyte in the adult may indicate an important role for this survival/antiapoptotic factor throughout germ cell development and maturation.

Carrier Proteins↗
PubMed2006

[Variability of human cytomegalovirus UL144 gene in low-passage clinical isolates analyzed by HMA-SSCP].

BACKGROUND: Human cytomegalovirus (HCMV) infection is an important infectious agent that results in neonatal disease and congenital deformity. HCMV infection may affect in many organs. The different symptoms and tissue tropism of HCMV infection perhaps resulted from the genetic polymorphism of HCMV. HCMV UL144 open reading frames encode a homologue of the tumor necrosis factor receptor. It seems important to study the strain-specific variability of UL144 sequence in low-passage clinical isolates and to discuss if the variability related to the clinical HCMV infection. METHODS: HCMV-UL144 gene was amplified by PCR assay in 65 low-passage clinical isolates and urine from 7 healthy children who were HCMV-DNA positive by quantitative PCR. All the positive PCR products were analyzed by Heteroduplex mobility assay and single-stranded conformation polymorphism (HMA-SSCP) and 32 of them were sequenced. RESULTS: Fifty-five isolates and 5 urine specimens were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in the UL144 ORFs. Comparing UL144 sequences and the corresponding symptoms showed that genotype 2 did not exist in megacolon isolates. And genotype 1 and 3 were the major types among microcephaly and jaundice isolates respectively. CONCLUSION: HCMV-UL144 existed in almost all the low passage isolates. HMA-SSCP assay is an easy and effective method to detect the genetype of HCMV-UL144 sequence. The characteristic of sequences in different isolates showed that UL144 gene may play an important role in HCMV infection.

Cytomegalovirus↗
PubMed2004

Recent approach for diagnosis of early HCV infection.

Detection of HCV infection during the window phase of infection, before seroconversion, is important in blood screening. However, a significant delay exists between the time of infection and the development of antibodies. The delay in window period can last up to 70 days. The aim of the present study was to investigate the kinetics of HCV markers during early infection, with detection of HCV core antigen as an early method for diagnosis. The study included determination of HCV RNA by qualitative and quantitative PCR, HCV core antigen detection by enzyme linked immunosorbent assay (ELISA) and specific serological markers including anti-HCV IgG and IgM. The study was carried out on 34 patients diagnosed as non A non B acute hepatitis and proved to be hepatitis C by qualitative HCV RNA PCR. Sixteen healthy control subjects were also included. From each consenting patient and control, blood samples were collected and serum was separated and subjected to determination of AST and ALT and the following virological laboratory tests: HCV core antigen detection by ELISA, determination of specific anti-HCV IgM and specific anti-HCV IgG, qualitative and quantitative determination of HCV RNA by second version of PCR. In patients, the median quantity of HCV RNA was 739.1 x 10(3) lu/ml with minimum quantity 2.1 x 10(3) lu/ml and maximum 38352.3 x 10(3) lu/ml. A comparison between the different diagnostic methods revealed that the highest sensitivity was for HCV-core antigen detection (82.4%), specificity was 100% negative predictive value was 72.2% and positive predictive value was 100%. Specific anti-HCV IgG had moderate levels of sensitivity (58.5%), specificity (75%), negative predictive value (46.2%)and positive predictive value (83.3%). The least sensitive method was the specific anti-HCV IgM (29.4%) with negative predictive value 40% but had specificity and positive predictive value of 100% of each. From this study we could conclude the followings: From virological methods, serological detection of specific IgM anti-HCV had the least sensitivity limits, while it had the highest specificity and positive predictive value. Specific anti-HCV IgG had moderate sensitivity and specificity. The most sensitive and specific tool for diagnosis of early HCV viraemia was the detection of HCV core Ag by ELISA when compared to molecular biological methods.

Adult↗
PubMed2006

RTPrimerDB: the real-time PCR primer and probe database, major update 2006.

The RTPrimerDB (http://medgen.ugent.be/rtprimerdb) project provides a freely accessible data retrieval system and an in silico assay evaluation pipeline for real-time quantitative PCR assays. Over the last year the number of user submitted assays has grown to 3500. Data conveyance from Entrez Gene by establishing an assay-to-gene relationship enables the addition of new primer assays for one of the 1.5 million different genes from 2300 species stored in the system. Easy access to the primer and probe data is possible by using multiple search criteria. Assay reports contain gene information, assay details (such as oligonucleotide sequences, detection chemistry and reaction conditions), publication information, users' experimental evaluation feedback and submitter's contact details. Gene expression assays are extended with a scalable assay viewer that provides detailed information on the alignment of primer and probe sequences on the known transcript variants of a gene, along with Single Nucleotide Polymorphisms (SNP) positions and peptide domain information. Furthermore, an mfold module is implemented to predict the secondary structure of the amplicon sequence, as this has been reported to impact the efficiency of the PCR. RTPrimerDB is also extended with an in silico analysis pipeline to streamline the evaluation of custom designed primer and probe sequences prior to ordering and experimental evaluation. In a secured environment, the pipeline performs automated BLAST specificity searches, mfold secondary structure prediction, SNP or plain sequence error identification, and graphical visualization of the aligned primer and probe sequences on the target gene.

Computer Graphics↗
PubMed2003

Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection.

Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log(10) unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.

Animals↗