Nested Reverse Transcriptase PCR Research Articles

Detection of porcine reproductive and respiratory syndrome virus (PRRS) genome fragments and anti-- bodies in chewing ropes allow -monitoring of PRRS in pig farms

Saliva samples from chewing ropes are a reliable diagnostic of porcine reproductive and respiratory syndrome virus (PRRSV) infections. The aim of this study was to test whether saliva samples taken with saliva swabs (cotton swabs and GenoTube Livestock) or with chewing ropes are suitable for monitoring PRRSV in unsuspicious farms, this means to detect a prevalence of 20% infected animals with a 95% probability. Saliva samples were collected from 12-16 pens in five pig farms by using a chewing rope for collective samples and by individual saliva swaps from five randomly selected animals per pen. A total of 291 animals from 58 pens in four study farms and 60 animals from 12 pens in one control farm were collected. The samples were taken from all age categories. According to the current monitoring system the analysis of five individual serum samples from the same pens served as the reference method for the relative sensitivity of the saliva samples. Serum and chewing rope samples were tested by ELISA for antibodies. Two different systems were used for the serum samples. Chewing ropes, saliva swabs (GenoTube Livestock) and serum samples were examined for virus genomes using a nested reverse-transcriptase PCR and a commercial real-time reverse-transcriptase PCR kit. Cohen's Kappa was used as a measure of agreement. PRRSV antibodies were detected in the chewing ropes of 44 pens and in the serum samples of only 34 pens. Viral RNA was found in 13 (chewing ropes), respectively 16 pens (serum samples). Saliva swabs (GenoTube Livestock) showed a lower relative sensitivity of 20.00% compared to serum samples. The agreement of the two serum analysis was very good for the ELISAs (κ = 0,911), and moderate for the PCR (κ = 0,706). The comparison of the chewing rope method with the analysis of the serum samples advocates this method as a suitable supplementary monitoring tool in PRRSV unsuspicious pig farms. Easy handling and lower examination costs of the chewing rope method allow higher testing frequency and would therefore improve the monitoring system. However, they are not an alternative to serum samples. Sampling with saliva swabs is unsuitable.

Direct detection of Trichomonas vaginalis virus in Trichomonas vaginalis positive clinical samples from the Netherlands

Trichomonas vaginalis is the most common sexually transmitted parasitical infection worldwide. T. vaginalis can carry a virus: Trichomonas vaginalis virus (TVV). To date, four TVV species have been described. Few studies have investigated TVV prevalence and its clinical importance. We have developed a nested reverse-transcriptase PCR, with novel, type specific primers to directly detect TVV RNA in T. vaginalis positive clinical samples.A total of 119T. vaginalis positive clinical samples were collected in Amsterdam and “s-Hertogenbosch, the Netherlands, from 2012 to 2016. For all samples T. vaginalis was genotyped using multi-locus sequence typing. The T. vaginalis positive samples segregated into a two-genotype population: type I (n=64) and type II (n=55). All were tested for TVV with the new TVV PCR. We detected 3 of the 4 TVV species. Sequencing of the amplified products showed high homology with published TVV genomes (82–100%). Half of the T. vaginalis clinical samples (n=60, 50.4%) were infected with one or more TVV species, with a preponderance for TVV infections in T. vaginalis type I (n=44, 73.3%). Clinical data was available for a subset of samples (n=34) and we observed an association between testing positive for (any) TVV and reporting urogenital symptoms (p=0.023). The nested RT-PCR allowed for direct detection of TVV in T. vaginalis positive clinical samples. This may be helpful in studies and clinical settings, since T. vaginalis disease and/or treatment outcome may be influenced by the protozoa”s virus.

Molecular detection of canine parainfluenza virus circulation among home-owned dogs in Shiraz, Iran

One of the major agents of infectious tracheobronchitis in dogs is canine parainfluinza virus (CPiV) which is known to cause acute-onset and highly transmissible cough. This study was aimed at determining the prevalence of CPI infection in dogs referred to the Veterinary Clinic of Shiraz University in 2013–2014. Nasal and tracheal swabs were taken from 30 apparently healthy dogs without respiratory signs and 50 sick dogs with clinical respiratory signs. Full set of characteristics including age, sex, breed, contact with other dogs, place of keeping, respiratory and general symptoms were recorded. The molecular detection of canine parainfluenza virus by semi nested reverse transcriptase PCR were performed and further confirmed by nucleotide sequencing of the last products. The results showed that the occurrence of CPiV infection in healthy dogs was 13.33% whereas it was 20% in dogs with respiratory signs. The sequencing and nucleotide blast showed no variation among determined sequences. The occurrence was higher in non-vaccinated and outdoor dogs. This study is the first attempt to detect CPiV infection in Fars province, Iran and the data obtained may be useful for outbreak preventions and to raise awareness of potential transmission of the disease to the small animals in the area.

Subclinical avian hepatitis E virus infection in layer flocks in the United States

The objective of this study was to determine patterns of avian HEV infection in naturally infected chicken farms. A total of 310 serum samples and 62 pooled fecal samples were collected from 62 chicken flocks on seven commercial in-line egg farms in the Midwestern United States and tested for avian HEV circulation. Serum samples were tested for the presence of anti-avian HEV IgY antibodies by a fluorescent microbead immunoassay (FMIA) which was developed for this study. The FMIA was validated using archived samples of chickens with known exposure (n = 96) and compared to the results obtained with an enzyme-linked immunosorbent assay (ELISA) based on the same capture antigen. There was an overall substantial agreement between the two assays (κ = 0.63) with earlier detection of positive chickens by the FMIA (P = 0.04). On the seven farms investigated, the overall prevalence of anti-avian HEV IgY antibodies in serum samples from commercial chickens was 44.8% (20–82% per farm). Fecal samples were tested for avian HEV RNA by a nested reverse-transcriptase PCR. The overall detection rate of avian HEV RNA in fecal samples was 62.9% (0–100% per farm). Sequencing analyses of partial helicase and capsid genes showed that different avian HEV genotype 2 strains were circulating within a farm. However, no correlation was found between avian HEV RNA detection and egg production, egg weight or mortality. In conclusion, avian HEV infection is widespread among clinically healthy laying hens in the United States.

Novel CT domain-encoding splice forms of CTGF/CCN2 are expressed in B-lineage acute lymphoblastic leukaemia

IntroductionConnective tissue growth factor (CTGF/CCN2) has been shown previously to be aberrantly expressed in a high proportion of paediatric precursor B cell acute lymphoblastic leukaemia (pre-B ALL), suggesting a potential oncogenic role in this tumour type. We therefore assessed CTGF mRNA transcript diversity in B-lineage ALL using primary patient specimens and cell lines. MethodsCTGF mRNA expression was evaluated by quantitative real-time PCR and Northern blotting. We performed a structural analysis of CTGF mRNA by nested reverse-transcriptase PCR and examined CTGF protein diversity by immunoblotting. ResultsNorthern blot analysis of pre-B ALL cell lines revealed short CTGF transcripts that were expressed in association with the active phase of cellular growth. Structural analysis confirmed the synthesis of several novel CTGF mRNA isoforms in B-lineage ALL cell lines that were uniformly characterised by the retention of the coding sequence for the C-terminal (CT) domain. One of these novel spliceforms was expressed in a majority (70%) of primary pre-B ALL patient specimens positive for canonical CTGF mRNA. Evidence that these alternative transcripts have coding potential was provided by cryptic CTGF proteins of predicted size detected by immunoblotting. ConclusionThis study identifies for the first time alternative splicing of the CTGF gene and shows that a short CTGF splice variant associated with cell proliferation is expressed in most cases of primary CTGF-positive pre-B ALL. This novel variant encoding only the CT domain may play a role in pre-B ALL tumorigenesis and/or progression.

Impact of host IL28B rs12979860, rs8099917 in interferon responsiveness and advanced liver disease in chronic genotype 3 hepatitis C patients.

Background and AimsGenetic polymorphisms near interleukin 28B gene are associated with spontaneous and treatment induced clearance of hepatitis C virus (HCV). Our objective was to evaluate the impact of interleukin 28B single nucleotide polymorphism (rs12979860, rs8099917) variability in HCV genotype 3 infected populations.Methods400 hepatitis C seroreactive patients from different population groups in Eastern and North Eastern part of India were assessed for host and viral genotypic analysis. 83 HCV genotype 3 infected patients were administered pegylated interferon- ribavirin therapy. Viral genotyping was performed using nested reverse transcriptase-PCR followed by direct sequencing methods. Host interleukin 28B genotyping was performed using real-time PCR based single nucleotide polymorphism analysis.ResultsOut of 400 hepatitis C seroreactive individuals, 73.25% were found to be RNA positive. HCV genotype 3 (65.87%) was found to be the major circulating strain in this region followed by genotype 1 (32.08%). rs12979860 CC genotype was significantly associated with sustained virological response in HCV genotype 3 infected population. In patients achieving rapid virological response, favourable CC/TT allele at rs12979860, rs8099917 was found to be predominant at both the alleles at 77%, 73.2% respectively; whereas in case of patients with relapsed HCV infection CT, TG alleles were found to be predominant. Additionally, CC genotypes at rs12979860 were found to be associated with sustained virological response in patients with high viral load (OR = 6.75, 0.05<p). HCV unfavourable rs12979860 TT, rs8099917 GG alleles were present in 34%, 27.6% patients with relapsed HCV infection respectively. Also unfavourable CT, TG genotypes were found to be predominant in patients with advanced stages of liver disease.ConclusionCC, TT the two favourable markers at SNPs rs12979860 and rs8099917 are strongly associated with sustained virological response in genotype 3 infected populations. This information will aid clinicians to effectively design response based treatment regimen.

The Use of Oral Fluid Samples Spotted on Filter Paper for the Detection of Measles Virus Using Nested RT‐PCR

Measles is the leading cause of death in infants, although a vaccine is available for its prevention. At this stage of measles elimination and eradication, it is so important to confirm clinically diagnosed measles cases in the laboratory but, developing countries have troubles in collecting and maintaining the cold chain of the specimens while transporting them to the laboratories. Therefore, filter papers are good candidates for simplification of specimen collection and transportation. In this research, the effects of the temperature, at which the dried specimens were kept, and the time duration the dried specimens were kept before being tested, were studied. Since there were not enough patients' oral fluid samples available, a nested reverse transcriptase PCR (RT-PCR) that detected measles virus (MV) from dried filter papers was set up using MV infected cells diluted in sterile phosphate-buffered saline (PBS). Dried specimens were stored at -25°C, 4°C, and room temperature for 1 day, 1, 2, and 3 weeks before being tested. This method was then applied to filter paper oral fluids collected from nine clinically diagnosed measles patients in Iran in 2010 which were tested after being kept at room temperature for 1 day, 1 and 3 weeks after preparation. The results showed that dried oral fluids on filter papers are reliable specimens for the detection of MV RNA using nested RT-PCR, but the nested RT-PCR results of low titer viruses dried onto filter papers are not reproducible and reliable.

Genetic Diversity of Avian Infectious Bronchitis Virus Isolated from Domestic Chicken Flocks and Coronaviruses from Feral Pigeons in Brazil Between 2003 and 2009

To detect the presence of infectious bronchitis virus or avian coronavirus, a nested reverse transcriptase PCR (RT-PCR) method was developed with the aim of amplifying a fragment of 530 bases, comprising the gene coding S1 protein. In the first step, all samples were submitted to RNA extraction, RT-PCR, and nested PCR. Next, only the positive nested-PCR samples were propagated in specific-pathogen-free (SPF) embryonated chicken eggs for virus isolation. Positive samples were then sequenced and analyzed using a molecular phylogeny approach. Tracheal swab samples were collected from 23 different domestic chickens distributed in three regions of Brazil, in the period between 2003 and 2009. Also analyzed were six swab samples (tracheal and cloacal) from asymptomatic pigeons (Columba livia), caught in an urbanized region in southeastern Brazil. The study revealed two major phylogenetic groups: one clustered with the Massachusetts vaccine serotype and another joined with the D207 strain. Interestingly, samples grouped with the Connecticut and Arkansas serotypes were also found. Pigeon isolates clustered with the Massachusetts serotype showed significant similarity (close to 100%) to those obtained from chickens. Only one pigeon isolate was seen to be grouped with the Connecticut serotype, and no correlation was observed between sample grouping and region origin. Understanding the diversity of genotypes and eco-epizootiology of the disease in different environments is expected to be helpful for vaccine production aimed at the main circulating variants. In this respect, one could also expect benefits in the management of other bird species that may act as avian coronavirus reservoirs.

Minimal Residual Disease Monitoring by Quantification of Fusion Gene Transcripts In Infant with MLL-rearranged Acute Lymphoblastic Leukemia Treated by MLL-Baby Protocol

AbstractAbstract 2731 Background.Despite many attempts worldwide treatment results for infants with MLL rearrangements and especially MLL-AF4 remain unsatisfied. MLL-Baby protocol was developed for infant acute lymphoblastic leukemia (ALL). In this treatment approach conventional chemotherapy is augmented by administration of all-trans retinoic acid (ATRA). This treatment program is successfully applied for infant ALL within Russian Federation and Republic of Belarus (L. Fechina et al, ASH 2007 #2828). Minimal residual disease (MRD) is a strong tool for risk-adapted treatment. Majority of infants carry MLL rearrangements, so in this group MRD monitoring by quantification of fusion gene transcripts (FGt) is fast, easy and cost-effective approach. Objective.To evaluate the prognostic significance of MRD monitoring by FGt measurements in MLL-rearranged infant ALL, enrolled into MLL-Baby study. Methods.Twenty three infants with defined MLL translocation partner genes who received at least two ATRA courses were included in the current study. Median of follow-up period in the observed group was 41 months. Presence of MLL rearrangements was detected by FISH and confirmed by long-distance inverse PCR (C. Meyer et al, 2005). MRD detection in bone marrow (BM) was performed by real-time quantitative PCR and qualitative nested reverse-transcriptase PCR as previously described (A. Borkhardt et al.,1994, J. van Dongen et al., 1999, N. Palisgaard et al., 1998, J. Gabert et al, 2003) with sensitivity 1E–05. MRD-negativity was defined as absence of FGt in both assays. Among 23 infants there were 13 MLL-AF4-positive patients (pts), 4 MLL-MLLT10-positive pts, 3 MLL-EPS15-positive pts, 2 MLL-MLLT1-positive pts and one MLL-MLLT3-positive patient. BM samples were obtained at the time of diagnosis, on day 15 of remission induction (time point (TP) 1), at the end of remission induction (TP2) and after each course of ATRA administration (TP3-TP9). Informed consent was obtained in all cases. Results.All patients were MRD-positive at TP1. At TP2 two MLL-MLLT10-positive patients became MRD-negative. At TP3 other 4 pts (3 MLL-AF4-positive and 1 MLL-MLLT1-positive) converted to MRD-negativity. By TP4 18 pts were MRD-negative, while FGt were detected in 5 pts. 2 pts became MRD-negative before protocol II (at TP9), while 3 pts never achieved MRD-negativity. Retrospectively, we compared prognostic significance of MRD at each TP. TP4 was the earliest TP when discriminative data was obtained. According to the qualitative MRD results at this TP pts were divided into MRD-positive and MRD-negative groups. The first group consisted of 18 pts with different MLL translocation partner genes, while the second group included 5 MLL-AF4-positive pts, who remained MRD-positive at TP4. Groups did not differ in age at diagnosis, sex distribution, initial WBC count, immunophenotype, type of MLL partner gene, number of blast cells at day 8 of dexamethasone prophase, BM status on day 15, CNS disease, and achievement of hematological remission at day 36. Number of relapses was significantly higher in the second group (p=0.017). Odds ratio was 20.00 with 95% CI 1.61–247.99. In the first group there were 3 relapses (in one MLL-AF4-positive case and two MLL-EPS15-positive cases) while in the second group 4 relapses occurred. Cumulative incidence of relapse for pts who achieved MRD-negativity by TP4 was 0.17, for MRD-positive pts 0.80 (p=0.005). 7-years event-free survival in the first group was 0.82±0,09, in the second group 0.20±0.17 (p=0.008) (fig 1). Conclusions.MRD monitoring by FGt measurements has significant prognostic value in infants with MLL-rearranged ALL treated by MLL-Baby protocol. In our series achievement of MRD-negativity by TP4 corresponds to favorable outcome in infant ALL with MLL rearrangements treated by MLL-Baby protocol. Persistence of MRD-positivity at TP4 allows to define group with high incidence of relapse. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Research on the association between Borna disease virus infection and the viral encephalitis

To investigate the infection of Borna disease virus (BDV) in unidentified viral encephalitis patients in Ningxia in order to explore if the nucleotide sequence and amino acid sequence in BDV p24 were homophylic with the overseas standard strain. We also intended to investigate the correlation between BDV infection and the unidentified viral encephalitis patients from Ningxia to lay an experimental basis for etiological diagnosis, prevention and treatment on certain human neuropsychiatric disorders. BDV p24 gene fragment was detected by nested reverse transcriptase polymerase chain reaction with fluorescence quantitative PCR (FQ-nRT-PCR) in peripheral blood mononuclear cells (PBMCs). Samples were from 59 unidentified viral encephalitis patients and 60 normal controls. For those positive products, gene sequence and amino acid sequence were then analyzed by BLAST and DNAsist 5.0. The positive rate of the BDV p24 gene fragment in PBMCs from the unidentified viral encephalitis (10.17%) was significantly higher than from the controls (0%) (P < 0.05). Data from the gene sequence on those positive products showed BDV p24 fragment in the patients with unidentified viral encephalitis from Ningxia was homophylic with strain H3915 detected from ill horses (97.67%), as well as with the strain H1766 (96.51%) and strain V (95.35%). However, their amino acid sequences remained the same. BDV infection might probably have existed in the unidentified viral encephalitis patients of Ningxia. The gene sequence seemed to be homophylic with that of standard strain H1766 and strain V, especially with strain H3915.

Detection and analysis of Borna disease virus in Chinese patients with neurological disorders

Borna disease virus (BDV) is a neurotropic RNA virus that is known to cause neurological disturbances in various animal species, potentially even humans. However, the association between BDV infection and human neurological disorders remains unclear. Between August 2005 and March 2006, 65 patients with neurological disorders were enrolled into our study. The presence of BDV p24 RNA from peripheral blood mononuclear cells (PBMCs) was investigated by using nested reverse transcriptase PCR (RT-PCR) assay. Borna disease virus p24 RNA was detected from PBMCs in six patients with viral encephalitis by using nested RT-PCR assay. However, BDV p24 RNA was not detected in patients with multiple sclerosis or peripheral nerve diseases. There might be possible associations between BDV infection and human viral encephalitis.

Disease‐Associated Novel CD46 Splicing Variants and Pathologic Bone Remodeling in Otosclerosis

Otosclerotic bone is supposed to show unique CD46 expression pattern because otosclerosis is an organ-specific disease with viral etiology. Otosclerosis is a complex bone remodeling disorder of the human otic capsule, which is associated with persisting measles virus infection. The general cellular receptor of measles virus is the CD46, which has 14-known splicing isoforms. Nucleic acid was extracted from ankylotic stapes footplates (N = 99) removed during stapedectomies. Consecutive histological, CD46 specific immunohistologic analysis, and multiple polymerase chain reaction (PCR) amplifications were performed. Measles virus was detected by seminested reverse transcriptase-PCR. Splicing variants of CD46 were identified by nested reverse transcriptase-PCR and finally determined by mass sequencing of complementary DNA. Measles virus RNA was detectable only in histologically otosclerotic stapes footplates. Virus negative-fixed stapes represent degenerative disorders of variable histopathology. Otosclerosis is featured by an increased number of osteoclasts showing strong CD46 immunoreaction in contrast to nonotosclerotic stapes fixations. Normal and nonotosclerotic stapes footplates show consistent expression of "c," "d," "e," "f," and "l" CD46 splicing isoforms. In contrast, four novel CD46 splicing variants were additionally detected in otosclerosis: os1, os2, os3, and os4. Newly described CD46 isoforms have shorter or missing transmembrane domain and a rare cytoplasmic tail with pathological or uncommon signal transduction; however, virus binding ability remains equal and invariable. These changes may be responsible for the smooth virus replication. A special expression pattern and altered functions of CD46 could explain the organ-specific and virus-associated pathogenesis of otosclerosis.

Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice

Cell-penetrating peptides (CPPs), containing arginine (R), 6-aminohexanoic acid (X), and/or beta-alanine (B) conjugated to phosphorodiamidate morpholino oligomers (PMOs), enhance their delivery in cell culture. In this study, the potency, functional biodistribution, and toxicity of these conjugates were evaluated in vivo, in EGFP-654 transgenic mice that ubiquitously express the aberrantly spliced EGFP-654 pre-mRNA reporter. Correct splicing and enhanced green fluorescence protein (EGFP) upregulation serve as a positive readout for peptide-PMO (PPMO) entry into cells and access to EGFP-654 pre-mRNA in the nucleus. Intraperitoneal injections of a series of PPMOs, A-N (12 mg/kg), administered once a day for four successive days resulted in splicing correction in numerous tissues. PPMO-B was highly potent in the heart, diaphragm, and quadriceps, which are key muscles in the treatment of Duchenne muscular dystrophy. We therefore investigated PPMO M23D-B, designed to force skipping of stop-codon containing dystrophin exon 23, in an mdx mouse model of the disease. Systemic delivery of M23D-B yielded persistent exon 23 skipping, yielding high and sustained dystrophin protein expression in body-wide muscles, including cardiac muscle, without detectable toxicity. The rescued dystrophin reduced serum creatinine kinase to near-wild-type levels, indicating improvement in muscle integrity. This is the first report of oligonucleotide-mediated exon skipping and dystrophin protein induction in the heart of treated animals.

Quantification of colorectal cancer micrometastases in lymph nodes by nested and real-time reverse transcriptase-PCR analysis for carcinoembryonic antigen.

Reverse-transcriptase PCR (RT-PCR) assays for carcinoembryonic antigen (CEA) have been described to identify lymph node micrometastases. These assays are not quantitative and can be confounded by false-positive results. The purpose of this study was to determine whether quantification of CEA in lymph nodes could more readily identify clinically relevant groups. Specimens included 400 lymph nodes from 64 patients undergoing colon resections. Specimens were tested by immunohistochemistry and by RT-PCR using nested primers for CEA. Specimens from 59 patients that were positive by nested RT-PCR were further quantified by detection of CEA mRNA fluorescence increase at a threshold PCR cycle. CEA was detected by nested RT-PCR analysis in 4 of 34 (12%) nodes of nonneoplastic disease, 2 of 13 (15%) nodes from T(1)N(0) patients, 32 of 81 (40%) nodes of T(2)N(0) patients, 49 of 109 (45%) nodes from T(3)N0 patients, and 92 of 163 (56%) nodes from T(1-4)N(1-2) patients. The overall presence of any RT-PCR-detectable CEA in nodes did not differentiate patient groups. Immunohistochemistry was positive in nodes from 7% of T(3)N(0) patients and 100% of T(1-3)N(1-2) patients. CEA quantification revealed that 0 of 7 patients with nonneoplastic disease and 2 of 17 (12%) patients with stage I T(1-2)N(0) cancers had one or more lymph nodes with >/=1.0 x 10(2) CEA transcripts per sample. In contrast, 4 of 13 (31%) patients with stage II T(3)N(0) cancer and 10 of 22 (45%) stage III patients with known metastases had lymph nodes with >/=1.0 x 10(2) CEA transcripts. These data suggest that quantification of CEA levels in lymph nodes may more accurately identify patients at risk for cancer recurrence than does routine nested RT-PCR or immunohistochemistry.

Identification of a novel bluetongue virus vector species of Culicoides in Sicily

The vectors of bluetongue virus are certain species of Culicoides biting midges, and in the Mediterranean area Culicoides imicola has long been considered to be the only field vector. In...

Deletion of the FHIT gene in human colorectal cancer is independent of high-risk HPV infection.

The fragile histidine triad (FHIT) gene, which is frequently lost in many cancers, has been identified as a candidate tumor suppressor gene at chromosome 3p locus 14.2. Human papillomaviruses (HPVs) are the major cause of cervical carcinoma and have been found to be able to integrate its genes into the chromosome 3 fragile site of cultured cells, deleting a piece of DNA which includes the FHIT gene. We used nested reverse transcriptase PCR and DNA sequencing to evaluate 32 cases of colorectal adenocarcinoma and matched nearby normal tissues for aberrant transcripts of FHIT and infection of high-risk HPVs. Aberrant transcripts of the FHIT gene were observed in 34.4% of colorectal adenocarcinoma and in 6.3% of matched nearby normal tissues. The HPV(16) DNA was detected in 21.9% of colorectal adenocarcinomas and 3.1% of matched nearby normal tissues using PCR. Only two cases of colorectal adenocarcinoma contained both aberrant transcripts of FHIT and HPV(16) infection. No HPV(18) infection was detected in the present study. Our results indicate that alteration of the FHIT gene and HPV(16) infection are important genetic events associated with colorectal adenocarcinoma. However, there is no correlation between FHIT abnormalities, clinicopathological features, and HPV(16) infection in colorectal adenocarcinoma.

Detection of Mycobacterium tuberculosis group organisms in human and mouse joint tissue by reverse transcriptase PCR: prevalence in diseased synovial tissue suggests lack of specific association with rheumatoid arthritis.

Infection with mycobacterial species, including Mycobacterium tuberculosis, has long been implicated in the etiopathology of rheumatoid arthritis (RA) on the basis of clinical and pathological similarities between tuberculosis and RA. Despite evidence of immune responses to mycobacterial antigens in RA patient synovial fluid, cross-reactivity between these and host joint antigens, and the presence of M. tuberculosis protein antigen in RA synovial fluid, a definite causal association with RA has not been shown. Previous studies from our laboratory using reverse transcriptase PCR (RT-PCR) of bacterial rRNAs have shown RA synovium to be colonized by a diverse range of bacteria, most of commensal origin. However, M. tuberculosis group organism (MTG) RNA sequences were found in one RA patient tissue. Since this was considered of sufficient interest to warrant further investigation, we devised a M. tuberculosis-specific nested RT-PCR test which could be used for detection of MTG in a mixed pool of bacterial crDNAs. This test was used to investigate the distribution of MTG in RA synovial tissue and also non-RA arthritis and healthy control tissues and was also used to examine the tissue distribution of MTG in an acute and chronic model of M. tuberculosis infection in the BALB/c mouse. MTG sequences were found in a high proportion of RA patient synovial tissues but also in non-RA arthritis control tissues at lower frequency. This likely reflects trafficking of persistent M. bovis BCG to inflamed joint tissue, irrespective of cause. MTG were not found in healthy synovial tissue or the tissue of patients with undifferentiated arthritis. In both the acute and chronic models of infection in BALB/c mice, M. tuberculosis was also found to have trafficked to joint tissues, however, no signs of inflammation, arthritis, or pathology associated with M. tuberculosis infection was seen. These combined results would argue against a specific causal role of MTG in RA-like arthritis; however, their role as adjuvant in immune dysfunction in an innately susceptible host cannot be excluded.

Gastroenteritis outbreaks associated with Norwalk-like viruses and their investigation by nested RT-PCR.

Norwalk-like viruses are the most common cause of gastroenteritis outbreaks and sporadic cases of vomiting and diarrhoea. In healthy individuals infection is often mild and short-lived but in debilitated patients infection can be severe. It is essential that the virus laboratory can offer a sensitive and specific test, delivered in a timely manner. We have developed a nested reverse transcriptase PCR based on published primers against the RNA polymerase gene and after comparison with electronmicroscopy used the assay to investigate 31 outbreaks of gastroenteritis. These were in diverse situations including nursing homes, small district hospitals, large general hospitals, a ferry ship, hotels, restaurants and staff canteens. A positive diagnosis was made in 30/31 outbreaks investigated giving an overall outbreak positive detection rate of 97%. At an individual patient level there was a positive diagnostic rate of 11.5% in a large hospital environment to 100% in smaller outbreak situations. The average patient positive rate was 34%. In addition we investigated 532 control faecal specimens from adults. Of these 530 were negative and 2 were repeatedly positive. It is essential that insensitive electronmicroscopy is replaced with the more sensitive reverse transcription PCR assays. These tests should be made available "on call" at weekends and public holidays. It is also important that outbreaks of NLV infection are monitored using sensitive RT-PCR assays so that the laboratory information can be used in ascertaining the spread and duration of the outbreak.

Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR.

A modified nested reverse transcriptase PCR (RT-PCR) method was used to detect the expression of nitrogenase genes in meso-oligotrophic Lake George, New York. Net (>20-microm pore size) plankton samples collected from two sites (Dome Island and Hague Marina) were extracted for total RNA and genomic DNA to determine the identity of diazotrophic organisms that were present and those that were actively expressing nitrogenase genes. Phylogenetic analysis of individual sequences cloned from PCR amplifications showed that there were phylogenetically diverse groups of bacteria that possessed a nifH gene, including representatives of unicellular and filamentous cyanobacteria, the alpha- and gamma-subdivisions of the division Proteobacteria (alpha- and gamma-proteobacteria), and a previously undefined group of bacteria. The phylotypes cloned from RT-PCR amplifications, which were actively expressing nifH transcripts, clustered with the unicellular and filamentous cyanobacteria, alpha-proteobacteria, and the novel bacterial cluster. No bacterial sequences were found which clustered with sequences from cluster II (alternative nitrogenases), III (nitrogenases in strict anaerobes), or IV (nifH-like sequences). These results indicate that there were several distinct groups of nitrogen-fixing microorganisms in the net plankton from both sampling sites and that most of the groups had representative phylotypes that were actively expressing nitrogenase genes.

A sensitive nested reverse transcriptase PCR assay to detect viable cells of the fish pathogen Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.).

A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between 1 and 10 cells. Total RNA was extracted from cultured bacteria, Atlantic salmon (Salmo salar L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR. In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable R. salmoninarum within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spawning to reduce vertical pathogen transmission.