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PubMed2026

Integrative multi-omics and machine learning identify the SPI1-METTL16-PLIN4 axis as a candidate driver of steatosis in HepG2 cells.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a prevalent metabolic disorder with limited therapeutic options. This study aimed to identify potential regulators and explore their functional roles in a cellular model of NAFLD. METHODS: WGCNA was performed on the hepatic transcriptomic dataset GSE126848 (31 NAFLD vs. 26 controls), followed by integration with serum proteomic data from 12 NAFLD patients and 12 healthy controls. Hub genes were prioritized using three machine learning algorithms. Functional validation was conducted in a HepG2 cellular steatosis model induced by high fructose (3.2 g/L) and oleic acid (400 μM) for 48 h. Lipid accumulation was assessed by Oil Red O staining and triglyceride/total cholesterol measurement. Inflammation was evaluated by TNF-α and IL-6 secretion (ELISA), and oxidative stress by ROS levels (flow cytometry). The binding interaction between METTL16 and PLIN4 mRNA was validated by RNA immunoprecipitation (RIP)-quantitative PCR. METTL16-mediated m6A modification of PLIN4 was assessed by Methylated RIP (MeRIP)-quantitative PCR. Transcriptional regulation of METTL16 by SPI1 was examined by chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. RESULTS: Integrative analysis identified PLIN4 as a core hub gene. PLIN4 was upregulated in the HepG2 steatosis model (P < 0.001). PLIN4 knockdown alleviated lipid droplet accumulation (P < 0.001), reduced TNF-α and IL-6 secretion (P < 0.01), and decreased ROS levels (P < 0.001) in fructose/oleic acid-treated HepG2 cells. Mechanistically, METTL16 mediated its m6A modification to enhance PLIN4 mRNA stability. Furthermore, SPI1 was found to transcriptionally activate METTL16 by binding to its promoter (P < 0.001). PLIN4 re-expression partially reversed the protective effects of SPI1 knockdown on lipid accumulation (P = 0.01), inflammation (P < 0.05), and oxidative stress (P < 0.001). CONCLUSION: This study identifies the SPI1/METTL16/PLIN4 axis as a potential regulatory mechanism contributing to in vitro steatosis, inflammation, and oxidative stress in steatotic HepG2 cells.

Humans↗

PubMed1997

Quantification of cytokine transcripts using polymerase chain reaction.

Quantitative PCR techniques are both attractive and daunting. Most users appreciate the need for accurate quantification, but achieving this is not always straightforward. It usually requires more technical equipment than classical PCR and common sense controls are no longer sufficient to generate confidence in the quantitative power of the assay. This review describes the principles that underlie the most commonly used quantitative PCR techniques and their different read-outs, and aims to provide the reader with the tools needed to discriminate between the different published techniques.

Animals↗

PubMed2004

Assessment of automated DNA extraction coupled with real-time PCR for measuring Epstein-Barr virus load in whole blood, peripheral mononuclear cells and plasma.

BACKGROUND: Epstein-Barr virus (EBV) DNA load monitoring in blood has been shown to be essential for the diagnosis of EBV-associated diseases. However, the methods currently used to assess EBV DNA load are often time-consuming and require prior blood separation. OBJECTIVES: The aim of this study was to evaluate the relative diagnostic value of EBV DNA load monitoring in whole blood, peripheral blood mononuclear cells (PBMCs) and plasma after automated DNA extraction using the MagNA Pure extractor followed by LightCycler real-time quantitative PCR (LC-PCR). STUDY DESIGN: First, EBV DNA load was assessed retrospectively after automated or manual extraction on 104 PBMC specimens. Second, EBV DNA load was determined prospectively with the automated extraction procedure in the whole blood, PBMCs and plasma of 100 samples from patients with EBV-related diseases (group 1, n = 20), HIV-seropositive individuals (group 2, n = 66), and healthy EBV carriers (group 3, n = 14). RESULTS: A good correlation was observed between automated and manual extraction on 104 PBMC specimens (r = 0.956; P < 0.0001). In the prospective study, 67 samples were positive in both whole blood and PBMCs, with a good correlation between EBV DNA loads in whole blood and PBMCs (r = 0.936; P < 0.0001). Only 18/100 samples were positive in plasma. Higher viral loads were regularly observed in the three blood compartments from group 1 than from groups 2 and 3. CONCLUSION: This study demonstrated that an automated extraction of EBV DNA is easier to perform in whole blood or plasma than in PBMCs and facilitates the standardisation of EBV DNA measurement by real-time quantitative PCR. The quantitative detection of EBV DNA load in whole blood appeared more sensitive than in plasma for infectious mononucleosis in immunocompetent patients, probably because of a rapid loss of plasmatic EBV DNA. In transplant patients, EBV DNA load monitoring in whole blood and in plasma turned out to be equivalent in terms of feasibility and accuracy for the early diagnosis of post-transplant lymphoproliferative diseases (PTLDs).

AIDS-Related Opportunistic Infections↗

PubMedDate not supplied

Comparative study of diagnosis of PD peritonitis by quantitative polymerase chain reaction for bacterial DNA vs culture methods.

INTRODUCTION: Peritonitis remains one of the main complications that afflict peritoneal dialysis patients. We conducted a pilot study to determine the feasibility and potential advantages of quantitative PCR (qPCR) assays for the presence of bacterial DNA in this clinical scenario. METHODS: 14 patients attending with 'cloudy bags' had PD fluid analyzed in accordance with Renal Association Standards. In addition, quantitative bacterial DNA analysis was performed on 50 mL samples of PD fluid. DNA was extracted using a Qiagen kit. Quantitative PCR assays using primers and probes targeted at 16S rDNA were used to measure the levels of bacterial DNA. Samples from 13 patients attending the department for other reasons served as negative controls. Laboratory staff were blinded to clinical details at the time of analysis. RESULTS: We determined a threshold of bacterial DNA whereby 11 of 13 negative controls were 'negative'. Significant bacterial DNA was found in 6 of 9 culture positive' peritonitis cases (p < 0.05 by chi(2). The 3 cases of 'no growth' peritonitis had 'insignificant' bacterial DNA. Serial DNA analysis was performed in 8 patients. Of the 6 patients who were 'cured' with standard antibiotic therapy, only 1 showed a rise in bacterial DNA from Day 1 to 5. But the 2 patients who relapsed after antibiotics had marked rises in bacterial DNA (p < 0.05 by chi(2). DISCUSSION: We showed that results from quantitative bacterial DNA PCR assays correlate with current microbiological tests despite the small size of this study. We also suggest that this technology might be clinically useful as an adjunct for cases of 'no growth' peritonitis and to identify those patients likely to relapse despite apparent clinical improvement with standard antibiotic therapy.

Bacteria↗

PubMed1999

Comparison of three PCR techniques for detecting cytomegalovirus (CMV) DNA in serum, detection of early antigen fluorescent foci and culture for the diagnosis of CMV infection.

Three PCR assays were developed for detection of cytomegalovirus (CMV) DNA in serum and were evaluated with samples from organ transplant recipients. The Qiamp Blood Kit was efficient for extraction of DNA from sera. Single-round PCR of a 293-bp region of CMV DNA was sensitive and highly specific for CMV targets and was more sensitive than detection of early antigen fluorescent foci (DEAFF) testing or isolation of CMV from buffy coat by cell culture. The results of a significant proportion of buffy coat samples were not interpretable because of toxicity in conventional culture or DEAFF tests. A non-competitive quantitative PCR test and semi-quantitative PCR test for the detection of CMV DNA in serum yielded comparable results for samples taken serially from three bone marrow transplant recipients. Single-round PCR was superior to conventional techniques for the diagnosis of CMV infection, was simple to perform and was completed rapidly. The semi-quantitative technique has added advantages where quantification is important.

Adult↗

PubMed2001

Evaluation of bioavailability of three types of IFNbeta in multiple sclerosis patients by a new quantitative-competitive-PCR method for MxA quantification.

Intracellular expression of human myxovirus protein A (MxA) is exclusively induced by type I IFNs (IFNalpha,beta,omega) or by some viruses and it is strongly increased under IFN treatment. We set up an internally controlled quantitative-competitive polymerase chain reaction (qc-PCR) that quantifies MxA mRNA expressed in human peripheral blood mononuclear cells (PBMC). Our qc-PCR is accurate because the mean ratio of copy number estimated by qc-PCR to that quantified spectrophotometrically is 1.08+/-0.03, moreover it is repeatable with high sensitivity (1 fg MxA/pg GAPDH). MxA mRNA was tested in 47 Relapsing-Remitting Multiple Sclerosis (RR-MS) untreated patients and in 48 patients treated with one of the 3 IFNbeta licensed for MS (24 with Rebif, 14 with Avonex and 10 with Betaferon). All the 48 treated patients were negative to IFNbeta neutralising antibodies (NABs) as tested in our laboratory using a cytopathic assay (CPE). MxA mRNA levels were detectable in all untreated patients (mean 24+/-18 fg MxA/pg GAPDH) and significantly higher levels were found in all the treated patients 12 h after IFNbeta administration (mean 499+/-325 fg MxA/pg GAPDH); furthermore, the three types of IFNbeta showed comparable bioavailability. Our data indicate that the bioavailability of the three available types of IFNbeta can be evaluated by MxA qc-PCR.

GTP-Binding Proteins↗

PubMed2006

Expression of endothelin receptors in frog, chicken, mouse and human pigment cells.

Several reports have shown the participation of vasoactive endothelins (ETs) in the regulation of vertebrate pigment cells. In the present study, we identified ET receptors in pigment cells of vertebrate species by RT-PCR assays, and compared the differential expression of the various subtypes in each species by quantitative PCR. RT-PCR was performed with specific primers for ETC, ETA(X) or ETA in Xenopus laevis melanophores, ETA or ETB(2) in chicken melanocytes, ETA or ETB in murine (B-16, S-91 or Melan-A) or human (SK-Mel 23 or SK-Mel 28) melanoma cells, and the products obtained were confirmed by cloning and sequencing. The results showed the presence of ETA(X), but not ETA mRNA, and confirmed the expression of ETC in X. laevis melanophores. ETA and ETB(2) mRNAs were also demonstrated in chicken melanocytes. ETA and ETB receptor were identified in S-91, B16 and Melan-A murine cells. In human melanoma cells, SK-Mel 23 and SK-Mel 28, we confirmed the presence of ETB mRNA, and also found ETA mRNA. The comparison between the two subtypes present in the pigment cell of each species and among species demonstrated that the expression of ETAs in chicken, mouse, and human melanocytes is negligible, as is the expression of ETA(X) in Xenopus melanophores. The relative expression, as determined by quantitative PCR, was as follows: chicken ETB>SK-Mel 23 ETB>S91 ETB>>>Xenopus ETC, suggesting that the endothelin system plays a major role in avian and mammalian pigment cell regulation, as compared to lower vertebrates. The phylogenetic analysis revealed that subtype A receptors were probably the most primitive ET receptors, directly deriving from the ancestral type; all the other receptors, B subtypes and C, originated from diverse derivative molecules.

Animals↗

PubMed2002

Enhanced force generation by corpus cavernosum smooth muscle in rabbits with partial bladder outlet obstruction.

PURPOSE: Growing clinical evidence suggests that benign prostatic hyperplasia induced partial bladder outlet obstruction is associated with an increased incidence of erectile dysfunction. We determined whether corpus cavernosum smooth muscle from rabbits with partial bladder outlet obstruction show any molecular or functional differences versus controls. MATERIALS AND METHODS: Force generation and relaxation of corpus cavernosum smooth muscle 2 weeks after partial bladder outlet obstruction by 125 mM. KCl, phenylephrine and field stimulation were determined. Expression of total smooth muscle myosin and alternatively spliced smooth muscle myosin isoforms were determined by reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative competitive RT-PCR, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Corpus cavernosum smooth muscle from sections of the penis were analyzed morphologically by immunofluorescence microscopy using antibodies to smooth muscle myosin and neurofilament protein. RESULTS: Corpus cavernosum smooth muscle from rabbits with partial bladder outlet obstruction generated 40% to 50% more force than that of sham operated rabbits in response to KCl or phenylephrine and was more difficult to relax. Although quantitative competitive RT-PCR and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that corpus cavernosum smooth muscle from rabbits with partial bladder outlet obstruction expressed only slightly more total smooth muscle myosin at the messenger RNA and protein levels, expression of the high adenosine triphosphatase isoform SM-B increased 2-fold. Morphological examination of corpus cavernosum smooth muscle sections revealed decreased innervation and increased smooth muscle bundle size. CONCLUSIONS: We present the novel finding of molecular and functional changes in the corpus cavernosum smooth muscle associated with partial bladder outlet obstruction. Although the inclusion of sham operation ruled out direct injury during surgery, a change in corpus cavernosum smooth muscle innervation induced by nerve compression by the ligature is likely to induce trophic changes in the corpus cavernosum smooth muscle leading to over expression of the SM-B smooth muscle myosin isoform, increased contractility and impaired relaxation.

Animals↗

PubMed2000

Quantitative analysis of the bidirectional fetomaternal transfer of nucleated cells and plasma DNA.

BACKGROUND: Recently, much interest has been generated on the fetomaternal transfer of nucleated cells and plasma DNA. However, there has been no systematic quantitative comparison of these two directions and two modalities of trafficking within the same study population. METHODS: The fetus-to-mother transfer of nucleated cells and plasma DNA in pregnant women carrying male babies was studied using a real-time quantitative PCR assay for the S:RY gene. For mother-to-fetus transfer, real-time quantitative PCR assays for the insertion/deletion polymorphisms involving the glutathione S:-transferase M1 and angiotensin-converting enzyme genes were used. RESULTS: Of the 50 informative mother-baby pairs, maternal DNA was detected in the cellular fraction of umbilical cord blood in 24% of cases (12 of 50), at a median fractional concentration of 2.6 x 10(-4) (interquartile range, 1.7 x 10(-4) to 3.6 x 10(-4)). In the plasma fraction of cord blood, maternal DNA was detected in 30% (15 of 50) of cases at a median fractional concentration of 3 x 10(-3) (interquartile range, 1 x 10(-3) to 1.6 x 10(-2)). For the other direction of trafficking, fetus-to-mother transfer of nucleated cells was detected in 26% of cases (13 of 50) at a median fractional concentration of 3.2 x 10(-4) (interquartile range, 0.6 x 10(-4) to 7.6 x 10(-4)). In the plasma fraction, fetal DNA was detected in 100% of maternal plasma (50 of 50) at a median fractional concentration of 3 x 10(-2) (interquartile range, 1.4 x 10(-2) to 5. 3 x 10(-2)). CONCLUSIONS: This study indicated that significantly more fetal DNA is present in the plasma of pregnant women compared with DNA from the cellular fraction of maternal blood. In addition, maternal DNA was demonstrated in both the cellular and plasma fractions of cord blood after delivery. This study has therefore determined the fundamental quantitative values for the bidirectional fetomaternal cellular and plasma DNA traffic.

Biological Transport↗

PubMed1999

Evaluation of different methods for the extraction of DNA from fungal conidia by quantitative competitive PCR analysis.

Five different DNA extraction methods were evaluated for their effectiveness in recovering PCR templates from the conidia of a series of fungal species often encountered in indoor air. The test organisms were Aspergillus versicolor, Penicillium chrysogenum, Stachybotrys chartarum, Cladosporium herbarum and Alternaria alternata. The extraction methods differed in their use of different cell lysis procedures. These included grinding in liquid nitrogen, grinding at ambient temperature, sonication, glass bead milling and freeze-thawing. DNA purification and recovery from the lysates were performed using a commercially available system based on the selective binding of nucleic acids to glass milk. A simple quantitative competitive polymerase chain reaction (QC-PCR) assay was developed for use in determining copy numbers of the internal transcribed spacer (ITS) regions of the ribosomal RNA operon (rDNA) in the total DNA extracts. These quantitative analyses demonstrated that the method using glass bead milling was most effective in recovering PCR templates from each of the different types of conidia both in terms of absolute copy numbers recovered and also in terms of lowest extract to extract variability. Calculations of average template copy yield per conidium in this study indicate that the bead milling method is sufficient to support the detection of less than ten conidia of each of the different organisms in a PCR assay.

DNA, Fungal↗

PubMed2006

Design and evaluation of 16S rRNA sequence based oligonucleotide probes for the detection and quantification of Comamonas testosteroni in mixed microbial communities.

BACKGROUND: The beta-proteobacterial species Comamonas testosteroni is capable of biotransformation and also biodegradation of a range of chemical compounds and thus potentially useful in chemical manufacturing and bioremediation. The ability to detect and quantify members of this species in mixed microbial communities thus may be desirable. RESULTS: We have designed an oligonucleotide probe for use in fluorescent in situ hybridization (FISH) and two pairs of PCR primers targeting a C. testosteroni subgroup. The FISH probe and one of the PCR primer pairs are suitable for quantification of C. testosteroni in mixed microbial communities using FISH followed by quantitative image analysis or real-time quantitative PCR, respectively. This has been shown by analysis of samples from an enrichment of activated sludge on testosterone resulting in an increase in abundance and finally isolation of C. testosteroni. Additionally, we have successfully used quantitative PCR to follow the C. testosteroni abundance during a laboratory scale wastewater bioaugmentation experiment. CONCLUSION: The oligonucleotides presented here provide a useful tool to study C. testosteroni population dynamics in mixed microbial communities.

Colony Count, Microbial↗

PubMed1996

A novel quantitative 'stretch PCR assay', that detects a dramatic increase in telomerase activity during the progression of myeloid leukemias.

Telomerase activation is important for carcinogenesis. However, the timing and magnitude of the activation during cancer development are unknown. In this study, a new PCR-based method for measuring telomerase activity was developed and shown to be very useful for quantitative analysis of human telomerase. Using this assay, blood or bone marrow cells from healthy donors, and patients with chronic myelogenous leukemia (CML) and acute myelogenous leukemia (AML) were examined as to their relative activity. Telomerase activity present in normal peripheral blood cells was generally very low. However, significant activity was detected occasionally in samples derived from younger healthy donors. Striking telomerase activation was observed at the time of the blastic crisis in CML: no samples from chronic phase cases showed significant activity, while all cases with a well established crisis showed strong activity. Most AML cases were telomerase-positive. Quantitative analyses revealed that the relative titer varied among the AML patients, from as low as found in normal cells to as high as found in cell lines. However, a tendency that the activity was higher in relapsed cases than in fresh ones was suggested. In summary, telomerase was activated during the progression of the clinical stages in leukemias. This observation suggests that shortened telomeres and increased telomerase activity might be necessary for cancer cells to undergo clonal evolution towards more malignant phenotypes in advanced stages.

Adult↗

PubMed2006

Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells.

In this chapter, we describe a gene-specific quantitative polymerase chain reaction (QPCR)-based assay for the measurement of DNA damage, using amplification of long DNA targets. This assay has been extensively used to measure the integrity of both nuclear and mitochondrial genomes exposed to different genotoxins, and has proved particularly valuable in identifying reactive oxygen species-mediated mitochondrial DNA (mtDNA) damage. QPCR can be used to quantify the formation of DNA damage, as well as the kinetics of damage removal. One of the main strengths of the assay is that it permits monitoring the integrity of mtDNA directly from total cellular DNA without the need for isolating mitochondria, or a separate step of mtDNA purification. Here we discuss advantages and limitations of using QPCR to assay DNA damage in mammalian cells. In addition, we give a detailed protocol for the QPCR assay that helps facilitate its successful deployment in any molecular biology laboratory.

Animals↗

PubMed2002

Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks.

Despite the clinical application of adeno-associated virus (AAV) gene therapy, the titration of viral stocks has not yet been standardized. This complicates the comparison of viral stocks between laboratories. Functional titering of AAV is time-consuming, requires the manipulation of hazardous material, and often has a high degree of variability. We established an optimized real-time quantitative polymerase chain reaction (RQ-PCR) titration assay to determine viral titers and compared it with a functional green fluorescent protein (GFP)-based titration method. With a combination of improved lysis procedures and RQ-PCR protocols we could decrease the intraexperimental coefficient of variation (CV) from 0.24 +/- 0.03 to 0.042 +/- 0.004 and the interexperimental CV from 0.34 +/- 0.06 to 0.093 +/- 0.028 following functional and RQPCR-based titration, respectively. This low variability conforms to even the strictest quality standards required, for example, in clinical laboratories. The highly standardized titration by RQPCR described here will be especially advantageous for groups working on AAV-based gene therapy in a good manufacturing practice setting.

Base Sequence↗

PubMed2004

[Quantitative analysis of SMN gene copies in spinal muscular atrophy].

OBJECTIVE: To study the genetic basis in the patients with clinical diagnosis of spinal muscular atrophy(SMA) but without survival motor neuron telomeric copy (SMN-T) deletion; the relationship between the SMN-C (centromeric) copies and the phenotype; and the distribution of SMN-C and SMN-T copies in the SMA patients, the carriers and the controls. METHODS: Quantitative PCR analysis of SMN-T and SMN-C copies were carried out in 45 patients, 25 consanguineous and 33 control individuals. The patients were identified by clinical manifestation and muscular pathology. Two internal standards of SMN-T and cystic fibrosis transmembrane conductance regulator (CFTR) were constructed. Nonradioactive and nonfluorescence-labelling competitive PCR were used. The numbers of SMN-T and SMN-C copies were determined by calculating the ratios of SMN-T/CFTR and SMN-C/CFTR. RESULTS: Quantitation of SMN-T gene copies in SMA patients revealed that nine cases of type I-III were homozygously deleted. Two cases of type III had only one copy and four cases of type III had two copies. SMA IV and other type cases had two copies. Nine cases of consanguineous individuals had one copy, but other 16 had two copies. All of the normal individuals had two copies. Analysis of SMN-C copies showed that SMA I had < or = 2 copies, II-III had < or = 3 copies, SMA IV and others had 0-3 copies, the consanguineous individuals and normal individuals had 0-3 copies. CONCLUSION: The number of copies determined by PCR quantitative assay of SMN-T is in accordance with the result of PCR qualitative assay of homozygous deletion. Quantitative assay of the number of copies can find out the cases and the carriers of heterozygous deletion. The SMA phenotype is related to the number of copies of SMN-C; the smaller the number of copies the patient has, the severer the patient's phenotype will be. The pathogenesis of SMA IV and other types of SMA may not relate to SMN gene.

Cyclic AMP Response Element-Binding Protein↗

PubMed2006

A new Real-Time-RT-PCR for quantitation of human endogenous retroviruses type K (HERV-K) RNA load in plasma samples: increased HERV-K RNA titers in HIV-1 patients with HAART non-suppressive regimens.

Viral components of the human endogenous retroviruses type K (HERV-K) have been largely detected in plasma from HIV-1 infected individuals. A Sybr Green Real-Time RT-PCR approach was optimized for detection and quantitation of HERV-K RNA titers in plasma samples using the iCycler technology. The method detected 1000 HERV-K RNA copies/mL of plasma sample. The Intra- and Inter-assay performance revealed a coefficient of variations that ranged from 0.2 to 2.46%, demonstrating accuracy and reproducibility. We quantified the HERV-K RNA load in 20 HIV-1 patients receiving highly active antiretroviral therapy (HAART). We found increased HERV-K RNA titers in patients with non-suppressive HAART (patients who may develop drug-resistance and/or received suboptimal therapeutic doses), compared to suppressive regimens (p < 0.001). HERV-K RNA was not detected in HCV-1 positive or seronegative controls. Sequencing of Real-Time RT-PCR products revealed particular HERV-K subtypes activated in the HIV-1 infection. The application of this assay could expand the understanding of the role of HERV-K in the HIV-1 infection and others pathological conditions.

Analysis of Variance↗

PubMedDate not supplied

Identification of patterns of transmission of Salmonella within swine production systems using pulsed field gel electrophoresis (PFGE) and repetitive sequence polymerase chain reaction (REP-PCR): a quantitative analysis.

Pulsed field gel electrophoresis (PFGE) using 3 enzymes (Spe I, Xba I, Avr II) and repetitive sequence polymerase chain reaction (REP-PCR) with 3 primers (BOX, ERIC, REP) were compared with respect to their validity as a method for identifying transmission of Salmonella on swine farms. Sixty-eight isolates of Salmonella were obtained from feces of swine, cats, mice, and birds, insect body parts, water and floor samples, and boot scrapings collected on 9 swine farms in Illinois USA. Genetic distances between isolates were calculated using the Dice matching coefficient. Cluster analysis of distance matrices was conducted using the UPG-MA algorithm. There was no significant difference between PFGE and REP-PCR in the genetic diversity detected; however, REP-PCR differentiated between 14 pairs of isolates which PFGE identified as identical. There were no significant differences between PFGE and REP-PCR in identifying all or most close genetic links as isolates from the same farm, the same building, and from the same sampling visit, suggesting ecological validity for both methods. Thus, REP-PCR should be considered as an acceptable and perhaps preferable alternative to PFGE as a genotyping method for studies of Salmonella transmission.

Animals↗

PubMed2000

Estimating viability of Cryptosporidium parvum oocysts using reverse transcriptase-polymerase chain reaction (RT-PCR) directed at mRNA encoding amyloglucosidase.

The purpose of the present study was to determine if reverse transcriptase-polymerase chain reaction (RT-PCR) directed at mRNA encoding the enzyme amyloglucosidase (CPAG) could serve as a indicator for C. parvum oocyst viability. Oocysts were stored for 1-11 months in the refrigerator and at monthly intervals extracted for total RNA for RT-PCR analysis. An aliquot of these C. parvum oocysts was inoculated into neonatal mice which were necropsied 4 days later for ileal tissue that was analyzed by semi-quantitative PCR to determine the level of parasite replication. The CPAG RT-PCR assay detected RNA from as few as 10(3) C. parvum oocysts. An effect of storage time on both RT-PCR signal and mouse infectivity was observed. RNA from oocysts stored for 1-7 months, unlike oocysts stored for 9 or 11 months, contained CPAG mRNA that was detectable by RT-PCR. A gradual decrease in the RT-PCR signal intensity was observed between 5 and 7 months storage. The intensity of RT-PCR product from oocysts and the signal from semi-quantitative PCR of ileal tissue DNA from mice infected with these same aged oocysts were comparable. The RT-PCR assay of CPAG mRNA in cultured cells infected with viable C. parvum oocysts first detected expression at 12 h with highest expression levels observed at 48 h post-infection. These results indicate that CPAG RT-PCR may be useful for differentiating viable from non-viable C. parvum oocysts and for studying the expression of the gene for amyloglucosidase in vitro.

Animals↗