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.     

Using polymerase chain reaction for rapid sex-determination of a case of sirenomelia

Developing endoluminal technology has permitted the management of selected aneurysms using stent-grafts, but the applicability and durability of these new devices has not yet been proven. Standard treatment of mycotic aneurysms generally requires arterial ligation, excision and debridement, and autologous or extraanatomic synthetic bypass. A saphenous vein-covered stent was used to exclude an expanding, mycotic, superficial femoral artery aneurysm in a critically ill patient. Although stent-graft exclusion was intended as a bridge to standard therapy, the mass resolved, the superficial femoral artery remains patent, and the patient is symptom-free at 1 year without further intervention. Additional experience is required to determine whether stent-grafts have a role in the management of mycotic aneurysms.     

Sex identification of turkey embryos using a multiplex polymerase chain reaction

A considerable portion of the W chromosome in Gallinaceous birds consists of tandem repetitive DNA. In the turkey, a 0.4-kb PstI element is repeated about 10,000 times in the female diploid genome but is undetectable as such a unit in males. In this study a multiplex polymerase chain reaction was developed to identify the sex of turkeys based upon the PstI repeat. The technique utilized two pairs of primers, the first pair was designed to amplify a region of the PstI repetitive element, resulting in the production of a 177-bp fragment in females. The other pair was designed to amplify a region of the adenosine triphosphate (ATP) synthase gene, present in both males and females. The simultaneous use of all four primers in the same reaction resulted in the coamplification of a 177-bp and a 250-bp fragment in females and a 250-bp fragment in males. This technique was used to verify the sex of 45 adults of known sex and to identify the sex of 74 embryos from Day 5 to hatch. This procedure is rapid and permits the sexing of many embryos in a short time. The ability to sex early embryos can facilitate studies on avian sex determination.     

A reverse transcriptase polymerase chain reaction assay for the detection of thermophilic Campylobacter spp

The purpose of this study was to determine the most appropriate method of normalisation for dual fine wire electromyography of shoulder muscles. Five healthy subjects were studied, with one muscle investigated in each subject (2 supraspinatus, 2 infraspinatus, 1 subscapularis). Three dual fine wire electrodes were inserted 1 cm apart around the recognised insertion points. Each subject performed five types of cyclic exercise on an isokinetic muscle dynamometer with an isometric maximal voluntary contraction (MVC) being performed before and after the exercise protocol. The EMG signal was normalised using each of the MVC voltage, the peak voltage and the whole-cycle mean voltage. There was a considerable difference (5-143%) between the MVC signals pre- and post-protocol, although no systematic trend was demonstrable. The overall mean between electrode variation in the normalised signal measured at the peak of the cycle ranged from 48-71% when normalised to pre-protocol MVC, but only 4-13% when normalised to the peak voltage and 9-17% using the whole-cycle mean voltage. However the pattern of activation within the movement cycle, which was preserved by normalisation using the peak or mean signal, was consistent between different electrode positions. It was concluded that the EMG signal depended on electrode position even when near the recognised insertion point, and that the MVC signal is highly variable in magnitude between electrodes and between pre- and post-protocol measurements.     

Detection of chlamydia trachomatis by polymerase chain reaction assay in nonbacterial prostatitis

BACKGROUND: Melasma is a common disorder of facial hyperpigmentation among Asian women. Many modalities of treatment are available but none is satisfactory. OBJECTIVE: This study was undertaken to see if glycolic acid peels are effective and safe in the treatment of melasma and fine facial wrinkling. METHODS: Ten Asian women with moderate to severe melasma were recruited into the study. The women had twice daily applications of a cream containing 10% glycolic acid and 2% hydroquinone (Neostrata AHA Age Spot and Skin Lightening Gel) to both sides of the face, and glycolic acid peels every 3 weeks (20-70%) to one-half of the face using Neostrata Skin Rejuvenation System. All patients had to use a sunblock (SPF 15%). At regular intervals and at the end of 26 weeks (or after eight peels) the degree of improvement of pigmentation and fine facial wrinkling on each side of the face were assessed. Any skin irritation or side effects were also noted. Assessment was by an independent dermatologist, the patients themselves, and the use of the Munsell color chart and photographs. The nonparametric Wilcoxon Rank-Sum test was used for statistical analysis. RESULTS: The melasma and fine facial wrinkling improved on both sides of the face. The side that received glycolic acid peels did better but the results were not statistically significant (P > 0.059). CONCLUSION: A cream containing 10% glycolic acid and 2% hydroquinone (Neostrata AHA Age Spot and Skin Lightening Gel) improved melasma and fine facial wrinkling in Asian women. In combination with glycolic acid peels at 3-week intervals the lightening of melasma is subjectively much better. This improvement does not reach statistical significance and the sample size is small (n = 10).     

Rapid diagnosis of tuberculous meningitis by polymerase chain reaction assay of cerebrospinal fluid

A polymerase chain reaction (PCR) method for the rapid diagnosis of tuberculous meningitis (TBM) was used to study prospectively 47 cerebrospinal fluid (CSF) samples from 45 patients. Twenty CSF samples were from patients with clinically suspected TBM and another 27 samples came from patients without clinically suspected TBM. Mycobacterial DNA was detected in 15 CSF samples (14 from patients with clinically suspected TBM and 1 from a patient not suspected of having TBM). Of the PCR-positive samples, 4 were also positive for mycobacterial culture. However, 32 PCR-negative samples were all culture-negative. All samples were negative for the acid-fast bacillus by direct smear. The single PCR-positive patient in the clinically unsuspected TBM group was initially diagnosed as suffering from aseptic meningitis on the basis of his clinical features. The mycobacterial culture of his CSF specimen was also positive and a revised diagnosis of an aseptic type of TBM was made. The estimations of specificity and sensitivity in this study were 100% and 70% respectively. The results showed that using a PCR to detect mycobacterial DNA in CSF for the early diagnosis of TBM is not only a rapid but also an accurate method.     

Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification

We describe a PCR-based assay for determining c-erbB-2 oncogene amplification in breast cancer in which we use the TaqMan system. Two fluorogenic probes anneal to the target between primers for c-erbB-2 and beta-globin genes and contain both a reporter dye (6-carboxy-fluorescein) and a quencher dye (6-carboxy-tetramethyl-rhodamine). During the extension phase of the PCR cycle, the 5'-->3' exonuclease activity of Taq polymerase cleaves the hybridized fluorogenic probe, resulting in an increase of fluorescence emission of the reporter dye that is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. Assay performance showed adequate precision and a lower detection limit and good correlation with the results obtained in the same samples by a competitive PCR assay (n = 25, r = 0.94, P < 0.01). This homogeneous assay is time-saving, avoids usually cumbersome postamplification procedures (that can be additional sources of inaccuracy and contamination), and seems suitable for determination of c-erbB-2 oncogene amplification in tumor specimens.     

Real-time detection of allele-specific polymerase chain reaction products by automated ultra-thin-layer agarose gel electrophoresis

Ultra-thin-layer agarose gel electrophoresis, a novel combination of agarose slab gel electrophoresis and capillary gel electrophoresis was introduced in conjunction with laser-induced fluorescence (LIF) scanning detection for the analysis of polymerase chain reaction (PCR) products. Allele-specific fragments, amplified from genomic DNA of patients with congenital adrenal hyperplasia (most often caused by mutations of 21-hydroxylase gene, CYP-21), were used as a model system to investigate the applicability, sensitivity and resolving power of the method. The allele-specific products were generated by PCR and separated by ultra-thin-layer agarose gel electrophoresis. The double-stranded DNA fragments were easily visualized in real-time via complexation during the separation process by the intercalator dye TO-PRO-3 which was part of the separation gel-buffer system. In this way, the migrating dsDNA-dye complexes were detected in real-time by a scanning LIF detection system with sub-nanogram sensitivity. The system employs a 632-nm solid-state laser and an avalanche photodiode detector scanning to the separation platform by means of a fiber bundle system. Automated ultra-thin-layer agarose gel electrophoresis with 'on the fly' TO-PRO-3 staining of dsDNA fragments and LIF detection system proved to be a very fast, high-throughput separation method for individual or multiplexed PCR products, with excellent sensitivity.     

Subtype-specific polymerase chain reaction for the identification of HIV-1 genetic subtypes circulating in Africa

The association between cancer and the increased tendency for bleeding and clotting is well established. Many investigators have reported on the interaction between tumor-related and plasma-related factors that may be responsible for this predisposition in cancer patients. The precise role of any of these factors in thrombosis or bleeding remains speculative. A good understanding of the course of the underlying malignancy, mechanisms of actions of the agents used for treatment, and a high degree of clinical suspicion are required to direct practicing physicians towards an objective and timely diagnosis of these complications. The goal of this article is to provide a broad overview of a few important hemostatic problems encountered in cancer patients.     

Species identification of intestinal microsporidiosis in HIV-positive patients using the polymerase chain reaction

BACKGROUND: Microsporidia are opportunistic parasites which, due to their morphologic characteristics, continue presenting diagnostic problems. Species-specific identification of microsporidia has become important because of varying levels of response to albendazole, which is the only effective treatment for some kinds of intestinal microsporidiosis. Although these parasites cause up to 50% of otherwise unexplained chronic diarrhea in HIV-positive patients, the number of reported cases is still very scarce in our country when compared to the existing HIV-positive population. METHODS: Intestinal microsporidiosis in HIV-positive patients with diarrhea was investigated using the modified trichrome staining technique. Microsporidia species identification was done by indirect immunofluorescence (IIF) and polymerase chain reaction (PCR) with specific primers. RESULTS: Six new cases of intestinal microsporidiosis caused by Enterocytozoon bieneusi were diagnosed in Madrid (Spain). All patients were in an advanced state of the HIV infection and they presented CD4+ values equal or inferior to 100 x 10(6)/I. CONCLUSIONS: Due to the number of cases that are accumulating, microsporidia must be included among the enteropathogens responsible for chronic diarrhea in HIV-positive individuals in Spain. The PCR technique using specific primers is a suitable determinator of the microsporidia species implicated in this intestinal pathology.     

A simple, rapid and sensitive detection of salmonella in food by polymerase chain reaction

A polymerase chain reaction (PCR) method was developed for detection of salmonella in food. A set of PCR primers was designed to amplify a 199 bp salmonella-specific DNA fragment derived from a repetitive DNA of Salmonella Weltevreden. The assay detected all 52 most prevalent serovars found in contaminated food in Thailand and no cross-reaction was observed with other non-salmonella organisms. The limit of detection was 1 fg of purified target DNA or five bacteria from pure culture. The detection of artificially contaminated food performed following a 6 h enrichment step was three bacteria per gram and the result was obtained within 4 h.     

Development of a rapid method for quantitative evaluation of Mycobacterium tuberculosis growth based on competitive polymerase chain reaction

A competitive polymerase chain reaction (cPCR) assay for the quantitative evaluation of Mycobacterium tuberculosis growth was developed based on co-amplification of genomic DNA and a modified DNA fragment derived from a well-conserved region of the 16S rRNA gene. There was a good correlation between the number of DNA copies in the sample, indicated by competitive PCR, and the number of colony forming units determined by conventional culture methods.     

Direct detection of Mycobacterium tuberculosis using polymerase chain reaction assay among patients with hepatic granuloma

BACKGROUND: In liver tuberculosis, demonstration of acid bacilli by conventional methods remains futile. Since the definitive diagnosis of liver tuberculosis is based on the histologic evidence of granulomatous process with caseation necrosis, seen in only a third of cases, the diagnosis is made retrospectively by response to empirical anti-tuberculous drug therapy. AIMS: Our objective is to establish a polymerase chain reaction assay for detection of Mycobacterium tuberculosis affecting the liver using the paraffin-embedded liver biopsy specimens obtained from patients with hepatic granulomas. METHODS: As positive control, patients having either "definitive" (n=8) or "presumptive" (n=9) tuberculosis on the basis of clinical, microbiological, histologic data and their positive response to empirical treatment of anti-tuberculous drugs were used. Patients with hepatic granulomas secondary to schistosomiasis (n=6), sarcoidosis (n=2) and other liver diseases (n=10) were used as negative control. RESULTS: Of those patients who were diagnosed as having "definitive" and "presumptive" liver tuberculosis, positivity by one-step polymerase chain reaction was 100% and 44%, respectively. Using the nested polymerase chain reaction, positivity increased to 78% with "presumptive" liver tuberculosis. In contrast, the polymerase chain reaction assays were negative among all patients with hepatic granuloma due to non-tuberculous-in-origin and other liver diseases. CONCLUSIONS: The overall positivity of this polymerase chain reaction assay (88%) compares favorably with that of other conventional methods (12%). Thus, this polymerase chain reaction assay may be a reliable diagnostic tool for liver tuberculosis in a patient population in which the prevalence of diseases associated with hepatic granuloma is common.     

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.     

Urinary inhibitors of polymerase chain reaction and ligase chain reaction and testing of multiple specimens may contribute to lower assay sensitivities for diagnosing Chlamydia trachomatis infected women

Clinical and morphologic features of a progressive polyneuropathy in young mature Alaskan Malamutes are described. Clinical signs included progressive paraparesis, synchronous pelvic limb gait, exercise intolerance, hyperesthesia, hyporeflexia, muscle atrophy, and tetraplegia. Electromyographic testing revealed diffuse fibrillation potentials and positive sharp waves in limb muscles, especially in muscles below the elbow and stifle. Pathologic findings in skeletal muscles and peripheral nerves included neurogenic muscle atrophy, focal or diffuse loss of myelinated nerve fibers, myelinoaxonal necrosis, and variable demyelination or remyelination. Ultrastructural changes included axonal degeneration, presence of numerous Büngner bands, and denervated Schwann cell subunits. The nature and distribution of abnormal electrophysiologic and pathologic findings were suggestive of a distal sensorimotor polyneuropathy, which we have termed idiopathic polyneuropathy of Alaskan Malamutes to distinguish this condition from hereditary polyneuropathy of Norwegian Alaskan Malamutes, last described in 1982.     

Species-specific identification of microsporidia in stool and intestinal biopsy specimens by the polymerase chain reaction

Horton temporal arteritis, or gigantocellular arteritis, is a panarteritis involving the mid-size and large arteries, in particular the temporal surface artery. This pathology is normally found in the elderly, particularly females. It can be manifest with some typical symptoms (i.e. cephalea, fever, visual disorders even leading to blindness mandibular claudication, high ESR, moderate anemia), there may be aspecific, atypical signs (i.e. only cephalea and fever, or widespread myalgia and artralgia) or it may arise following a cerebro-vascular accident. Treatment of this form of arteritis is based on the use of high doses of corticosteroids over a long period of time (at least 1-2 years). The present work describes a surgical technique for biopsying the temporal artery. The technique consists of withdrawal of a segment of the artery from the main branch and the frontal branch of the temporal surface artery. Since this form of arteritis often presents segmentary lesions, it is advisable to take a 4-5 cm sample so as to prevent false negatives. This simple surgical procedure can be performed under local anesthesia and is practically complication-free. The authors then discuss the indications for temporal artery biopsy and report 3 clinical cases (case no. 1 is an example of the classical manifestation while cases no. 2 and 3 are atypical, aspecific forms). Since both the classical and atypical forms of gigantocellular arteritis require high doses of corticosteriods over a long period of time, the temporal artery biopsy procedure is highly useful in formulating an accurate diagnosis. The well known side effects to long-term cortisone use make it necessary to use all the available instruments in making the correct diagnosis. Bilateral biopsy can be performed in those cases where the first biopsy proved negative but gigantocellular arteritis is still suspected.