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PubMed2002

mRNA-specific reverse transcription-polymerase chain reaction from human tissue extracts.

Reverse transcription-polymerase chain reaction (RT-PCR) has become the method of choice for detection of mRNA transcripts, including those of low abundance obtained from small precious samples of human tissue. A major confounding problem for standard reverse-transcription-priming strategies is the presence of contaminating genomic DNA (gDNA) carried over from the original "RNA" extract into the RT and PCR steps. The contaminating gDNA contains a processed pseudogene sequence-which lacks introns but contains a poly(A) tail-for commonly studied internal reference genes beta-actin and GAPDH, and target genes GSTM1, GSTP1, and others. These pseudogene sequences therefore confound standard-design "RNA-specific" PCR primer pairs which rely, for cDNA versus gDNA specificity, on the pair-spanning introns, or one of the individual primer oligos spanning an exon/exon splice site, because these features are lacking in processed pseudogene sequences. The result is false RT-PCR positives for these "housekeeper" genes in total RNA extracts; the gDNA processed pseudogene is mistaken for mRNA gene transcript. A universal RT primer has been designed that targets the poly(A) tail of mRNA and adds a unique tag sequence not otherwise existing in the human genome. Genomic DNA does not incorporate this RT-inserted unique tag. PCR is then performed using a transcript-specific forward primer and a reverse primer that is identical to the unique tag incorporated at RT. Only cDNA made with this RT primer is compatible with this reverse PCR primer, thus eliminating confounding signal from contaminating gDNA. This method performs RNA-specific qualitative and quantitative evaluation of gene expression, while preserving the sensitivity of standard RT-PCR techniques. Applications to low-copy transcripts in human samples are demonstrated.

Cell Extracts↗

PubMed2002

The patterns of accumulation of cellular RNAs in cells infected with a wild-type and a mutant herpes simplex virus 1 lacking the virion host shutoff gene.

Cellular RNA extracted from quiescent human foreskin fibroblasts harvested at 1, 3, 7, or 12 h after infection was profiled on Affymetrix HG-U95Av2 arrays designed to detect 12,626 unique human transcripts. We also profiled RNA extracted from cells harvested at 1 and 7 h after infection with a mutant lacking the gene (DeltaU(L)41) encoding a protein (vhs) brought into cells by the virus and responsible for nonselective degradation of RNA early in infection. We report the following: (i) of the 12 tested genes, up-regulated at least 3-fold relative to the values of mock infected cells, 9 were confirmed by real-time PCR. The microchip assays analyses indicate that there were 475 genes up-regulated > or =3-fold. The up-regulated genes were clustered into 15 groups with respect to temporal pattern of transcript accumulation, and classified into 20 groups on the basis of their function. The preponderance of cellular genes up-regulated early in infection play a predominant role in transcription, whereas those up-regulated at later times respond to intracellular stress or concern themselves with the cell cycle and apoptosis. (ii) The number of genes up-regulated early in infection was higher in cells infected with the DeltaU(L)41 mutant. Conversely, more genes were down-regulated late in infection with wild-type virus than with mutant viruses. Both observations are compatible with the known function of the U(L)41 gene product early in infection and with degradation of cellular RNAs in the absence of replenishment by de novo transcription of cellular genes.

Cells, Cultured↗

PubMed1987

Insulin-like growth factor I gene expression in GH3 rat pituitary cells: messenger ribonucleic acid content, immunocytochemistry, and secretion.

Insulin-like growth factor I (IGF-I) is present in multiple tissues and cell types. Expression of the IGF-I gene was examined in GH3 cells, a rat pituitary tumor cell line secreting GH and PRL. Increasing concentrations of RNA extracts of GH3 cells yielded a linear increase in hybridization intensity with a 32P-labeled mouse IGF-I cDNA probe. Northern analysis of GH3 cells poly(A) RNA revealed IGF-I mRNA transcripts 1.3, 5.3, and 7.7 kilobases in size. Poly(A) RNA extracts of BALBc/3T3 fibroblasts, a cell line dependent on exogenous somatomedins for DNA synthesis, and of JEG-3 cells, a choriocarcinoma cell line, did not hybridize with the IGF-I cDNA probe. GH3 cells showed positive immunoperoxidase staining using a rabbit anti[Thr59]IGF-I antibody which was largely blocked by prior incubation of the antibody with excess IGF-I. Negligible background peroxidase activity was present in cells incubated with a rabbit nonimmune serum and PBS. Furthermore, BALBc/3T3 fibroblasts showed only weak specific staining with the IGF-I antibody. Finally, GH3 cells secreted IGF-I into the culture medium in a time-dependent fashion, while neither 3T3 nor JEG-3 cells produced detectable medium levels of the peptide after 72 h of incubation. As IGF-I is known to inhibit GH production by the pituitary, the data shown suggest that locally produced IGF-I may regulate GH secretion in an autocrine or paracrine fashion.

Animals↗

PubMed1997

Type I insulin-like growth factor receptor gene expression in normal human breast tissue treated with oestrogen and progesterone.

The epithelial proliferation of normal human breast tissue xenografts implanted into athymic nude mice is significantly increased from basal levels by oestradiol (E2), but not progesterone (Pg) treatment at serum concentrations similar to those observed in the luteal phase of the human menstrual cycle. Type I IGF receptor (IGFR-I) mRNA and protein have been shown to be up-regulated by E2 in MCF-7 breast cancer cells in vitro in which IGF-I and E2 act synergistically to stimulate proliferation. We have investigated the expression of the IGFR-I mRNA in normal human breast xenografts treated with or without E2 or Pg alone and in combination. Northern analysis of 20 micrograms of RNA extracted from the breast xenograft samples showed no hybridization with 32P-labelled IGFR-I probe, although an 11-kb species of IGFR-I mRNA could be seen when 20 micrograms of RNA extracted from either MCF-7 breast cancer cells or human breast carcinomas was examined in this way. In order to analyse the expression of IGFR-I mRNA in breast xenografts, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) was employed in which RNA loading, reverse transcription and PCR efficiencies were internally controlled. The data indicate that the IGFR-I mRNA is up-regulated by two to threefold compared with untreated levels by 7 and 14 days E2 treatment. In contrast, 7 or 14 days Pg treatment down-regulates the receptor mRNA to approximately half that of untreated levels, whereas combination E2 and Pg treatment produced a twofold increase in IGFR-I mRNA levels compared with untreated tissue. The results are consistent with the suggestion that E2 may act to stimulate proliferation indirectly via a paracrine mechanism involving IGFs in normal as well as malignant human breast epithelial cells.

Animals↗

PubMed2005

The optimization of quantitative reverse transcription PCR for verification of cDNA microarray data.

cDNA microarray analysis is highly useful for monitoring genome-wide changes in gene expression that occur in biological processes. Current standards require that microarray observations be verified by quantitative (Q)-PCR or other techniques. Few studies have optimized Q-PCR for verification of microarray findings. The current study assessed several variables affecting Q-PCR fidelity, including RNA extraction methods, mRNA enrichment, primers for reverse transcription, and cDNA amplification detection methods. Also assessed was the choice of reference gene on which other gene expression changes are based. The RNA for ribosomal protein S28 was found to be ideal for this purpose, with minimal variance in expression among isogenic drug-resistant cell lines. We also found that oligo (dT) primers were superior to random hexamers and that RNA extracted by the RNeasy method gave consistent S28 gene amplification without the need for mRNA enrichment, particularly when TaqMan probes were used. Nevertheless, sensitivity was sufficiently high with SYBR Green I that it was the preferred, least costly method for amplification product detection, even for low-abundance transcripts. Using the optimal method, 91-95% of the differences in gene expression identified between the cell lines by cDNA microarray analysis could be confirmed by Q-PCR, significantly superior to previously described methods.

Antibiotics, Antineoplastic↗

PubMed1986

Expression of the myc cellular proto-oncogene in human thyroid tissue.

The expression of cellular oncogenes was examined in human thyroid tissue. Four thyroid adenomas and three thyroid carcinomas expressed c-myc oncogene mRNA transcripts, which were not expressed in normal thyroid tissues. Agarose gel electrophoresis of poly(A) RNA extracts of adenoma tissues followed by hybridization with v-myc DNA yielded two distinct c-myc mRNA species [2.1 and 4.0 kilobases (kb)]. Thyroid cancer tissue poly(A) RNA revealed a predominant larger c-myc mRNA species (approximately 6 kb) and a smaller 2.1-kb species. After enzymatic dispersion of thyroid adenoma cells, cytoplasmic dot blot hybridization of immobilized thyroid adenoma RNA extracts with the v-myc cDNA probe showed stimulation of c-myc mRNA expression by TSH in a dose- and time-dependent manner. After 72-h pretreatment with serum-free medium, the quiescent adenoma cells yielded a negligible hybridization signal which was stimulated by TSH (10 mU/ml) after 6 h of treatment. Although normal cultured thyroid cells did not contain detectable c-myc mRNA, TSH also stimulated the myc protooncogene expression in a time-dependent manner in normal thyroid cells. These results demonstrate the expression of c-myc mRNA in human thyroid tumors and the in vitro induction of c-myc mRNA in both normal and adenoma thyroid cell cultures by TSH.

Adenocarcinoma, Papillary↗

PubMed1998

Quantification of insulin-like growth factor-1 (IGF-1) mRNA: modulation of growth intensity by feeding results in inter- and intra-tissue-specific differences of IGF-1 mRNA expression in steers.

The effect of constant and compensating body growth velocities on IGF-1 mRNA expression was studied in various tissues of growing steers. Twenty-six steers were allocated to three groups in which the average daily gains were kept either constantly high on intensive feeding, low on pasture feeding or were accelerated to compensatory growth after feed restriction. All animals were slaughtered at 570+/-2.6 kg and samples were collected from liver, heart, kidney and from 4 different muscles (m. splenius, m. soleus, m. cutaneus truncii and m. semispinalis capitis), which were selected in order to include maximal differences in fibre composition as well as in growth impetus. IGF-1 mRNA was quantified by a validated internally standardised RT-PCR method. The amount of RNA extracted from the various tissues investigated was constant within each type of tissue and showed no differences between treatment groups. As indicated by a constant ratio between the amount of RNA extracted and the DNA concentrations, there was no effect of the feeding on total transcriptional activity. The order of IGF-1 mRNA abundance per g tissue was liver > > kidney > heart > skeletal muscle. The different feeding regimen resulted in significant differences of IGF-1 mRNA expression rates in all organs showing different patterns between organs. IGF-1 mRNA concentrations showed muscle specific differences and also divergent reactions in response to the differing growth rates. These results support that the liver is the main IGF-1 producing tissue; above that they indicate that skeletal muscle, in particular when taking its absolute mass into account, might considerably contribute to the IGF-1 levels in blood. Our findings demonstrate that IGF-1 mRNA expression is regulated tissue specifically not only between different organs but also within musculature.

Animals↗

PubMed1993

RNA from normal anterior endoderm/mesoderm-conditioned medium stimulates myofibrillogenesis in developing mutant axolotl hearts.

In the axolotl, Ambystoma mexicanum, a recessive cardiac lethal mutation causes an incomplete differentiation of the myocardium. Mutant hearts do not contain sarcomeric myofibrils nor do they beat. We have previously shown that normal anterior endoderm, medium conditioned by endoderm, or total RNA extracted from endoderm stimulates differentiation of mutant hearts in culture as indicated by the presence of organized myofibrils and rhythmic contractions of the "rescued" mutant heart tube. In this study, to get a more highly purified sample of the "active" molecule, RNA extracted from endoderm-conditioned medium and was assayed for its ability to promote myofibrillogenesis in mutant hearts. Mutant heart mesoderm responded to conditioned-medium RNA in a dose-dependent manner. Proteinase K treatment of the RNA did not affect inductive activity, while digestion with RNase A completely abolished the ability to rescue mutant hearts. Confocal laser scanning microscopy of immunostained, organ-cultured hearts revealed that mutant hearts contain reduced amounts of the sarcomeric protein tropomyosin in an amorphous distribution, whereas normal and corrected mutant hearts contain tropomyosin primarily in organized myofibrils.

Ambystoma↗

PubMed1975

[Radiobiological studies on the behavior of normal and tumorous plant tissues cultivated in vitro].

Vegetable tumoral tissue are more radiosensitive than the homologous normal tissue. Tumoral tissue are more stimulated by yeast extract and yeast s-ARN than normal tissue. Yeast extracts and s-RNA extracts from yeast also restore more the growth of tumorous tissue than normal tissue, which were subjected to gamma radiation from 60Co. A mixture of mononucleotides obtained from fractionated s-RNA has the same stimulatory effect. It is suggested that the active principle from yeast and s-RNA is a cytokinin like substance, which have been detected in yeast extract and in soluble ribonucleic acid from yeast.

Cobalt Radioisotopes↗

PubMed1993

Enhanced type III collagen gene expression during bleomycin induced lung fibrosis.

BACKGROUND: Intratracheal instillation of bleomycin into mice leads to deposition of collagen in the lung and fibrosis, but the mechanism for this is poorly understood. Enhanced collagen gene expression, increased collagen synthesis, decreased collagen degradation, and proliferation of fibroblasts have all been proposed as possible contributors. To obtain information on the activity of collagen producing cells at an early stage in the development of pulmonary fibrosis in situ hybridisation was used to detect and localise products of the type III procollagen gene. In addition, assay of type III procollagen gene expression was performed using dot-blot analysis of lung RNA extracts. METHODS: Lung fibrosis was induced in mice by intratracheal instillation of bleomycin sulphate (6 mg/kg body weight) and tissues were examined after three, 10, 21 and 35 days. RNA-RNA hybridisation was accomplished with riboprobes labelled with sulphur-35 which were generated from a 1.7 kb mouse procollagen a1(III) cDNA. In situ hybridisation was performed on sections fixed in paraformaldehyde and embedded in paraffin wax and steady state values of type III procollagen mRNA were assayed by dot-blot analysis of total lung RNA extracted by guanidium isothiocyanate. RESULTS: Data obtained using both techniques suggest that type III procollagen gene expression was enhanced in bleomycin induced fibrosis and that expression was maximal between 10 and 35 days after a single dose of bleomycin. The most active cells were located in interstitial areas around the conducting airways, although these cells were usually seen in areas with no histological evidence of fibrosis. Regions with the most advanced fibrosis, as assessed by histological methods, rarely contained cells with activity above the threshold detectable by this technique. CONCLUSIONS: These results suggest that activation of interstitial fibroblasts, with enhanced type III collagen gene expression, forms at least part of the mechanism leading to increased collagen deposition in bleomycin induced fibrosis and that this occurs before fibrosis is detected by conventional histological staining.

Animals↗

PubMed1995

Expression of novobiocin resistance genes in the novobiocin-producing organism Streptomyces niveus.

RNA isolated at intervals during fermentation from the novobiocin-producing wild-type strain of Streptomyces niveus and from a series of novobiocin-non-producing (Nov-) mutants was hybridized to DNA probes containing sequences which specify novobiocin resistance. The probes were made from inserts contained in the clones pGL101 and pGL103 which increase the level of novobiocin resistance of S. lividans transformants from 10 micrograms ml-1 to 50 micrograms ml-1 and 150 micrograms ml-1, respectively. No hybridization was detected with the pGL101 probe. The pGL103 probe hybridized to RNA extracted during the later stages of growth--a pattern corresponding to the transition from low to high level novobiocin resistance during growth of S. niveus wild-type cultures. Neither probe hybridized to RNA extracted from four Nov- mutants. These mutants showed variable levels of novobiocin resistance but none expressed the high wild-type levels. The authors conclude that expression of the DNA sequence in pGL103 is associated with high level novobiocin resistance.

DNA, Bacterial↗

PubMedDate not supplied

[Influence of a xylanase feed additive on Lactobacillus species in the jejunum of piglets].

rRNA extracts from the jejunum of rearing piglets fed a wheat/rye based diet with or without a xylanase feed additive were hybridized against eight Lactobacillus spp. specific digoxigenin labelled oligonucleotide probes. Known concentrations of sample- or culture RNA extracts were used to calibrate and quantify hybridization signals from different sample extracts and hybridizations. The xylanase feed additive tended to increase total eubacterial rRNA in the terminal jejunum, probably a result of increased Lactobacillus spp. rRNA in the central and terminal jejunum. However, individual animal variations were high. L. reuteri was the major Lactobacillus species followed by L. acidophilus and L. mucosae. L. amylophilus and L. crispatus were not detected in all RNA extracts. The results of this study show that the xylanase feed additive modifies Lactobacillus spp. populations on the species level in piglets in addition to their well known effects on digesta viscosity and nutrient digestibility. The implications of these effects for the intestinal microbiota require further analysis.

Animal Feed↗

PubMed1976

[RNA metabolism in chick embryo skin].

Explants from 7, 8, 9, 11, 13-day chick embryonic skin incorporating (3H) Uridine for different periods 1 hr, 3 or 4 hr and a chase with actinomycin) are studied with respect to free (F) or membrane bound (B) cytoplasmic polysomes and to RNA extracted from them. Polysome specific activity decreases at older stages but the amount of polysomes increases due to increased protein synthesis. At each stage B polysomes are less abundant but more radioactive than F polysomes. RNA extracted from each kind is analysed on sucrose gradients: one half of each fraction is precipitated by TCA to estimate total radioactivity, the other is retained on millipore at high salt concentration to estimate radioactivity in messenger-like RNAs due to their poly-A sequences. The pattern of the labelling of the different fractions of RNA changes with the length of incorporation, the stages of explants and the kind of polysomes (F or B); at 11-13 days the incorporation is slow, radioactivity is low and distributed among several peaks of poly-A RNA; at 7-8 days the incorportion is rapid, dispersed throughout the gradient; at 9 days, a midway stage, incorporation is particularly high into 12S and 24S fractions from B RNA. In the 5 studied stages the labelling of this 12S occurs early, remains for a longer time and cannot be chased. These observations suggest stability of the 12S RNA. Since, in 14-day chick embryos, feather keratin m RNA has been shown to sediment at 12S and although our experiments have been done with total skin because this differentiating tissue is the site of extensive interactions between dermis and epidermis, they suggest that this 12S RNA is the actual keratin m RNA and might be synthesised some days before the onset of keratin synthesis. Its template ability will be investigated at earlier stages.

Animals↗

PubMed1999

Multicenter quality assessment of PCR methods for detection of enteroviruses.

We conducted a multicenter evaluation of commercial and in-house PCR methods for the detection of enteroviruses. Three coded panels of test and control RNA samples, artificial clinical specimens, and representative enterovirus serotypes were used to assess amplification methods, RNA extraction methods, and reactivities with different enterovirus serotypes. Despite several differences between PCR methods, there was good agreement, although some variation in sensitivity was observed. Most PCR methods were able to detect enterovirus RNA derived from 0.01 50% tissue culture infective dose (TCID50) and were able to detect at least 1 TCID50 of enterovirus in cerebrospinal fluid, stool, or throat swab specimens. Most were also able to detect a wide range of enterovirus serotypes, although serotypic identification was not possible. Some laboratories experienced false-positive results due to PCR contamination, which appeared to result mainly from cross-contamination of specimens during RNA extraction. Provided that this problem is overcome, these PCR methods will prove to be a sensitive and rapid alternative to cell culture for the diagnosis of enterovirus infection.

Base Sequence↗

PubMed2002

Automated screening of blood donations for hepatitis C virus RNA using the Qiagen BioRobot 9604 and the Roche COBAS HCV Amplicor assay.

BACKGROUND AND OBJECTIVES: In order to reduce the potential for transmission of hepatitis C virus (HCV) from an RNA-positive, anti-HCV-negative blood donation, the National Blood Service (NBS) introduced nucleic acid amplification technology (NAT) testing for HCV in England and Wales. The objective of this study was to develop an automated assay using commercial components for the detection of HCV RNA in blood donations for transfusion. MATERIALS AND METHODS: The Qiagen QIAamp 96 'Viral RNA' and 'Virus' BioRobot kits for HCV RNA extraction, and the Roche COBAS HCV Amplicor v2.0 and AmpliScreen v2.0 assays for polymerase chain reaction (PCR) amplification and detection, were investigated. RESULTS: QIAamp technology and the BioRobot 9604 allow automation of the viral RNA extraction process. By combining the automated silica-membrane based QIAamp 96 Virus extraction and automated reverse transcription-polymerase chain reaction (RT-PCR) set-up with COBAS HCV AmpliScreen v2.0 amplification and detection it is possible to achieve a 95% detection level for HCV of 12.8 IU/ml. Cross-contamination studies have shown that use of the BioRobot 9604 does not pose a detectable contamination risk. Between 1999 and 2001, approximately 6.8 x 106 donations were tested in England and Wales, of which only four were found to contain RNA without anti-HCV. CONCLUSIONS: This combination of methods results in an assay with a high sample throughput, little 'hands-on' time and fast turnaround time that is also sufficiently sensitive to allow testing of pools of up to 96 samples at a time. These methods have been successfully introduced into routine use within the NBS for release of blood components with a shelf-life of longer than 24 h.

Blood Donors↗

PubMed1994

Differentially regulated immediate early genes in the rat uterus.

Estrogen stimulates cellular proliferation in the luminal epithelium, stroma, and smooth muscle of immature rat uterus. Progesterone administered concurrently with estrogen blocks the stimulatory effect of estrogen specifically in the epithelium, whereas progesterone administered alone stimulates proliferation in the endometrial stroma and myometrium. The present studies determined the effects of estrogen and progesterone on expression of the growth-associated, immediate early genes c-fos, c-jun, and jun-B in the luminal epithelium of the uterus. Hormonal effects were quantitated by Northern analysis of RNA extracted directly from the uterine luminal epithelium. Estrogen stimulated c-fos and jun-B expression, but repressed c-jun mRNA levels in the epithelium. In contrast, when whole organ RNA extracts were analyzed, estrogen increased mRNA levels for all three genes. Although progesterone administered alone showed no effect on mRNA levels in either epithelial or whole uterus extracts, it did attenuate the estrogen-induced increase in c-fos mRNA by 50% in whole uterus extracts and by 23% in epithelial extracts. The estrogen-induced increase in epithelial jun-B mRNA was not affected by progesterone pretreatment. Thus, in the immature rat uterus, no simple correlation exists between cellular proliferation and increased expression of the genes studied. However, progesterone completely blocked the repressive effect of estrogen on epithelial c-jun, suggesting a link between decreased c-jun expression and induction of cell proliferation in the uterine luminal epithelium. Estrogen repression of epithelial c-jun expression was hormone specific and sensitive to antiestrogen blockade. After estrogen treatment, epithelial c-jun mRNA decreased with a rate similar to its half-life, as determined in primary cultures of rat uterine cells. These results suggest that estrogen, acting through its receptor, directly represses transcription of c-jun in the uterine epithelium. Differences in hormonal regulation of immediate early genes between epithelial and nonepithelial uterine tissues probably results from tissue-specific transactivating factors that control the expression of these genes.

Animals↗

PubMed1969

RNA synthesis in cells infected with herpes simplex virus. II. Evidence that a class of viral mRNA is derived from a high molecular weight precursor synthesized in the nucleus.

Viral RNA extracted from the cytoplasmic polyribosomes of human epidermoid carcinoma no. 2 cells infected with herpes simplex virus (mRNA) had a sedimentation coefficient between 10S and 20S while that from nuclei of infected cells varied in size from 10S to >80S. Estimates of the maximum molecular weight of viral RNA from its sedimentation coefficients suggest that at least 10 per cent of the viral genome is transcribed as a single molecule. The ratio of RNA of different sizes found in the nuclei of cells pulse labeled for 12 minutes was approximately the same as those found in cells labeled for longer intervals implying that either some classes of viral mRNA were made as small molecules or that the large viral RNA molecules were cleaved soon after synthesis. Cytoplasmic mRNA competed to a level of at least 80 per cent in viral DNA-RNA hybridization tests with >50S RNA extracted from nuclei of infected cells. This is consistent with the hypothesis that viral mRNA is produced by cleavage of a large precursor RNA molecule.

Carcinoma, Squamous Cell↗

PubMed2005

Development of a new microwell hybridization assay and an internal control RNA for the detection of porcine noroviruses and sapoviruses by reverse transcription-PCR.

Recently, genetically diverse porcine noroviruses (NoV) and sapoviruses (SaV) were identified from field pig fecal samples. Reverse transcription (RT)-PCR is the primary method used for detection of human NoVs and SaVs. However, RT-PCR inhibitors frequently cause false-negative results. In this study, a competitive internal control (IC) RNA, specific for use in the SaV RT-PCR assay, was developed to monitor inhibition of RT-PCR; primers for detection of genetically diverse porcine NoVs and SaVs were designed; and microwell hybridization assays to confirm the specific RT-PCR products were developed. The primer pairs and the RT-PCR-hybridization combinations were compared using representative porcine NoV and SaV strains, positive pig fecal samples and a panel of 30 field pig fecal samples. Extracted RNA from 3 of 30 samples failed to amplify the IC RNA. However, this inhibition was not present after a 10-fold dilution of the extracted RNA. The five different RT-PCR-hybridization combinations developed specifically detected all three genotypes of porcine NoVs, all GIII porcine SaVs, unclassified JJ681-like, QW19 and LL26-like porcine SaVs, respectively. These RT-PCR-hybridization assays are specific, less time consuming and economical and particularly applicable to testing large number of samples for porcine NoVs and SaVs.

Animals↗