Rapid detection of Shiga-like toxin gene in feces with rapid-cycle polymerase chain reaction

A rapid detection for Shiga-like toxin in feces was developed with the nucleic acid extraction method by silicondioxide-guanidine thiothianate and rapid-cycle polymerase chain reaction by RapidCycler (model 1002; Idaho Technology, RC-PCR here after). Twenty-two fecal samples that were collected from patients with diarrhoea caused by E. coli O157:H7 and frozen for 6 months were examined directly by RC-PCR, conventional PCR assay using by ThermalCycler 9600-R (Roche, TC-PCR here after) and by the culture method using tellurite-cefixime sorbitol MacConkey (direct method). These examinations were done also after being injected into TCV-TSB and incutated at 35 degrees C overnight (indirect method). The sensitivity of RC-PCR and TC-PCR using a diluted suspension of broth enriched at 35 degrees C overnight were 4.1 pg and 410 fg, respectively. Positive results in the direct method were obtained in 7 for RC-PCR, 10 for TC-PCR and 5 for culture. Positive results on indirect assay were obtained in 9 for RC-PCR, 9 for TC-PCR and 7 for culture. It was demonstrated that the RC-PCR assay was able to detect Shiga-like toxin gene in feces in less than 90 minutes after being received at the laboratory.     

Confirmation of presumptive Salmonella colonies by the polymerase chain reaction

The potential of a genus-specific polymerase chain reaction (PCR) for the confirmation of Salmonella colonies was evaluated on 209 presumptive Salmonella colonies obtained by the standard method ISO 6579. The PCR method employing primers ST11 and ST15 (S. Aabo et al., Mol. Cell. Probes 7:171-178, 1993) gave results identical (100%) to those of the biochemical and serological identification, in terms of discrimination of Salmonella from non-Salmonella strains. PCR could be used directly on the colonies from selective plating media, which allowed a reduction of the time required for confirmation to a maximum of 6 h.     

Detection of Salmonella enteritidis in eggs by the polymerase chain reaction

A polymerase chain reaction (PCR) for the specific detection of the gene sequence, sefA, encoded by all isolates of Salmonella enteritidis, was developed. The PCR could detect as few as four S enteritidis washed bacterial cells but egg contents inhibited the PCR. Eggs spiked with 50 S enteritidis bacterial cells were homogenised, inoculated into buffered peptone water and grown at 37 degrees C for 16 hours, when the PCR was successful. A positive internal control was developed to differentiate between true and false negative PCR results for the detection of S enteritidis. In a limited trial of the egg handling procedures and the PCR, one of 250 chickens' eggs from retail outlets was found to be contaminated with S enteritidis.     

A simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces

A polymerase chain reaction (PCR) assay is described for the detection of parvovirus in feces of dogs and cats. A touch-down protocol was used which enabled the specific amplification of virion DNA from feces after a fast and simple boiling pretreatment. The sensitivity of PCR was as high as ten infectious particles per reaction which corresponds to a titer of about 10(3) infectious particles per gram of unprocessed feces. This renders the PCR about 10- to 100-fold more sensitive than electron microscopy, the standard method for parvovirus diagnosis. The very rapid and simple sample preparation recommends this PCR assay as an alternative technique for routine parvovirus diagnosis.     

Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction

A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella typhi in the blood specimens from patients with typhoid fever. Two pairs of oligonucleotide primers were designed to amplify a 343-bp fragment of the flagellin gene of S. typhi. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization by using a 32P-labeled 40-base probe internal to the amplified DNA. The nested PCR with two pairs of primers could detect 10 organisms of S. typhi as determined by serial dilutions of DNA from S. typhi. The peripheral mononuclear cells from 11 of 12 patients with typhoid fever confirmed by blood culture were positive for DNA fragment of the flagellin gene of S. typhi, whereas 10 blood specimens of patients with other febrile diseases were negative. With the nested PCR, S. typhi DNAs were detected from blood specimens of four patients with suspected typhoid fever on the basis of clinical features but with negative cultures. We suggest that the PCR technique could be used as a novel diagnostic method of typhoid fever, particularly in culture-negative cases.     

Detection of Helicobacter pylori gene by means of immunomagnetic separation-based polymerase chain reaction in feces

BACKGROUND: Detection of Helicobacter pylori is usually performed by culture, polymerase chain reaction (PCR), histology, or urease test on gastric biopsy samples. Although methods based on feces are non-invasive, their sensitivity has been relatively low. In this study, to improve its sensitivity, immunomagnetic separation (IMS) was used as a pre-PCR step for direct detection of H. pylori in feces. METHODS: Fresh fecal samples were taken from 72 patients attending for endoscopy. Of these, 57 patients had a positive H. pylori status according to the results of culture, histology, and PCR on gastric biopsy samples. Anti-H. pylori antibody-sensitized immunomagnetic beads were used to concentrate the bacteria. PCR was then performed to detect the H. pylori urease A-encoding gene. RESULTS: Of the 57 H. pylori-positive patients, 35 (61.4%) had positive fecal samples by IMS-based PCR method. None of the 15 H. pylori-negative patients had positive fecal samples. The sensitivity of this method was 61.4%, and the specificity 100.0%. CONCLUSIONS: This study confirms that non-invasive diagnosis of H. pylori infection could be made from feces by using IMS-based PCR.     

Application of the polymerase chain reaction to the epidemiological study of amoebiasis

Fresh gastric carcinoma specimens from 17 cases were collected. The neuroendocrine (NE) cells in gastric carcinoma (GC) and gastric mucosa adjacent to the carcinoma (GMAC) were observed using in situ hybridization of chromogranin A (CgA) oligonucleotide probe and compared with immunohistochemistry of CgA antibody. 5 of the 17 cases of GC showed CgA mRNA positive expression and 7 of the 17 cases expressed CgA protein positively. The simultaneous expression of mRNA and protein of CgA was present in 4 cases. The NE cells in GC not only can store and secrete CgA protein products but also possess the ability to synthesize NE products from gene expression.     

The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities

This article provides an overview of a new theory of suggested involuntariness in hypnosis, developed in conjunction with Irving Kirsch. The theory is based on the following ideas. First, high hypnotizable participants enter hypnosis with a conscious intention to feel and behave in line with suggested experiences and movements. Second, people who are easily hypnotized hold firm expectations that they will succeed in following the suggestions of the hypnotist. Third, the intention and expectation in turn function as response sets in the sense that they trigger the hypnotic response automatically. Fourth, given the intention to feel and behave in line with the hypnotist's suggestions, hypnotized individuals show no hesitation to experience the suggested movements as involuntary because (a) these movements are actually triggered automatically, and (b) the intention to cooperate with the hypnotist as well as the expectation to be able to do so create a heightened readiness to experience these actions as involuntary.     

Detection of Salmonella cells within 24 to 26 hours in poultry samples with the polymerase chain reaction BAX system

The parents of 470 students randomly selected from 1321 students attending a state high school were surveyed during the 1993-94 measles epidemic, by means of a take-home questionnaire. The response rate was 87%. Thirty stated that their child had measles during this epidemic; nine of these 30 gave a history of previous vaccination. Overall, 312 of the 470 (76%) stated that their child had been vaccinated, but only 34% indicated that they had vaccination records. There were no measles cases during this epidemic in the group with records. Those not vaccinated were at 10 times increased risk of contracting measles compared to those who had been vaccinated with or without records. Vaccine efficacy estimated in general a decade after vaccination based on parental recall of vaccination status regardless of whether they had vaccination records or not was 91% (95% CI 80%-96%). This calculation excluded 123 who claimed to have had measles prior to 1993 and 30 uncertain of their vaccination status.     

Application of the polymerase chain reaction for the detection of Ehrlichia canis in tissues of dogs

Doxycycline treatment resulted in a carrier status in 3 dogs and complete clearance of Ehrlichia canis in 2 dogs (Iqbal and Rikihisa, 1994). Using specimens obtained during that study applicability of polymerase chain reactions (PCRs) in detecting E. canis DNA in tissue specimens and correlation of PCR results with our previous cell culture isolation results were evaluated. PCRs using a pair of primers specific to E. canis 16SrRNA gene sequence were used to detect DNA of E. canis in tissues of 5 experimentally-infected dogs 2 months after doxycycline treatment. An approximately 600 bp product defined by the specific primers was amplified in blood, kidney, lymph nodes, liver, and/or spleen of 3 dogs from which E. canis was reisolated in cell culture. In contrast, E. canis DNA was not detected in tissue or blood specimens of the 2 dogs from which E. canis was not reisolated after doxycycline treatment or in 2 control uninfected dogs. The findings indicate PCR is effective in detecting E. canis in tissues.     

RNA extraction from virus-diseased sweet potato for reverse transcriptase polymerase chain reaction analysis

Apoptosis occurs in both clinical and experimental alcoholic liver disease. The mechanisms involved in alcohol-induced apoptosis of liver cells are not completely understood. Induction of cytochrome P450 2E1, the alcohol-inducible cytochrome P450, is one of the proposed mechanisms. Exposure of Hep G2 cells expressing cytochrome P450 2E1 to arachidonic acid leads to increased lipid peroxidation and apoptosis. Increased levels of iron in the liver also promote lipid peroxidation and are associated with increased numbers of apoptotic hepatocytes. Tumor necrosis factor (TNF) acting through its receptors can induce apoptosis in hepatocytes. Increased levels of tumor necrosis factor and its receptors have been described in alcoholic liver disease. The liver is also CD95 receptor positive and in liver tissue from patients with alcoholic hepatitis, the CD95 ligand is expressed at high levels in hepatocytes. Cytotoxic T lymphocytes could, through the CD95 receptor-ligand interaction, promote apoptosis.     

Value of the polymerase chain reaction for the detection of Toxoplasma gondii in cerebrospinal fluid from patients with AIDS

To investigate whether the polymerase chain reaction (PCR) on the BI gene of Toxoplasma gondii could contribute to the diagnosis of cerebral toxoplasmosis in patients with AIDS, we retrospectively tested CSF samples from 20 patients with AIDS suspected of having cerebral toxoplasmosis for the presence of T. gondii. Suspicion of cerebral toxoplasmosis was based on accepted criteria. Nine patients with AIDS with IgG antibodies to T. gondii but who were not suspected of having cerebral toxoplasmosis and four patients with AIDS seronegative for T. gondii served as negative control patients. T. gondii was demonstrated by PCR in the CSF from 13 of the 20 patients with AIDS suspected of having cerebral toxoplasmosis but was not demonstrated in the CSF samples from the nine control patients seropositive for T. gondii and the four control patients seronegative for T. gondii. The data were statistically evaluated. This study shows the value of PCR for the detection of T. gondii in CSF for the diagnosis of cerebral toxoplasmosis in patients with AIDS.     

Helicobacter pylori detection by polymerase chain reaction in gastric juice and its correlation with the histology (Giemsa)]

HP infection is involved in the pathogenesis of several gastroduodenal diseases, as type B chronic gastritis, duodenal and gastric ulcer, MALT lymphoma and gastric cancer. The recent availability of molecular techniques, specifically the PCR, allow us to detect very low amounts of the bacterium. The aim of the study is to evaluate the presence of HP in gastric juice by PCR technique and to correlate this findings with histology (Giemsa) of gastric mucosa. Gastric juice PCR positive findings were found in 10/31 (32.3%) HP positive patients at histology. We concluded that HP in gastric juice is possible to detect by molecular techniques. In our study 32.3% of the patients showed the presence of HP in gastric juice.     

Detection of Francisella tularensis by the polymerase chain reaction

A 27-year-old woman and a 13-year-old girl diagnosed with juvenile dermatomyositis in childhood developed clinical findings of partial lipodystrophy 10 years after diagnosis. Exhaustive clinical and laboratory examinations showed an association with other abnormalities: hypertrichosis, steatohepatitis, and an abnormal insulin response to the glucose loading test in the first patient. Hypertrichosis, steatohepatitis, insulin-resistant diabetes mellitus, and acanthosis nigricans were observed in the second patient. Renal function was normal in both patients. Although a localized form of lipodystrophy has been reported associated with connective tissue disease (connective tissue lipoatrophy), the partial form has been infrequently described in association with juvenile dermatomyositis.     

Application of interspersed repetitive sequence polymerase chain reaction for construction of yeast artificial chromosome contigs

This work is devoted to the comprehension of the sorption mechanism of uranyl ions on chitosan particle dispersions. The uranyl concentration measurements were obtained by inductively coupled plasma atomic emission spectrometry (ICP-AES) and we considered the role of various physicochemical parameters (pH; nature and concentration of added salts; degree of acetylation, DA). The use of appropriate calculation software allowed us to determine the chemical nature of uranyl species in solution in relation to these different parameters. The optimal pH of fixation has been found to be within 6.5-7.5 and can be related to the necessity of having both deprotonated amino groups and no carbonate ions, which are a strong complexant of uranyl ions, thus inhibiting their interaction with chitosan. The decrease of metal uptake with an increase of DA and the lack of influence of ionic strength, confirm the results obtained with pH and allowed us to suppose the formation of a complex with chitosan amino groups rather than interactions of an electrostatic nature.     

Studies on inactivation of hepatitis B virus with polymerase chain reaction

Polymerase chain reaction-ethidium bromide (PCR-EB) method for detecting hepatitis B virus DNA (HBV-DNA) was established with good specificity and a detection limit of 1 pg HBV-DNA, minimum HBV infection dose in susceptible animal, chimpanzees, could be detected with it. Determination of inactivation of HBV-DNA could be inactivated with active chlorine 1,250 mg/L for 60 minutes, or 2,500 mg/L for 30 minutes, 10 pg HBV-DNA in purified Dane particles could be inactivated by active chlorine 625 mg/L for 10 minutes. Accordingly, use of PCR to evaluate the effects of chlorine disinfectant in inactivating HBV was feasible, and HBV-DNA was a more reliable index for inactivation of HBV than HBsAg.     

Comparison of polymerase chain reaction with culture and enzyme immunoassay for diagnosis of pertussis

The risk for human infection with Lyme disease appears linked to the abundance of infected vector ticks, principally Ixodes dammini Spielman, Clifford, Piesman & Corwin, in the eastern United States. Habitat destruction by burning, although not well studied, has long been considered as an effective alternative to synthetic insecticides as a means of reducing tick populations. We evaluated the effect of a single spring burning of the woodland understory on the transmission risk of Lyme disease spirochetes (Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner) on Shelter Island, Long Island, NY. Following a burn in early April 1991, the abundance of nymphal I. dammini was 49% lower in the burned portion of a woodlot compared with the unburned portion. However, risk of encountering nymphs infected with B. burgdorferi remained similar in both burned and unburned woods. It is suggested that burning vegetation may disproportionately kill deer-derived rather than rodent-derived nymphs, significantly reducing tick abundance without affecting transmission risk.     

Positive identification of the specificity of polymerase chain reaction product

A method of positive identification of the specificity of polymerase chain reaction (PCR) product using internal oligonucleotide probe is introduced. The hybridization was done on the agarose gel which was dried after electrophoresis. Detection of the expression of T cell receptor a chain variable (TCR V alpha) genes on mRNA level was used as the experimental model. Twenty nine TCR V alpha gene subfamilies could be distinguished clearly in healthy human peripheral blood lymphocytes by this method. Positive identification of PCR product on dried agarose gel by internal oligonucleotide probe is relatively simple and less time consuming.