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.     

Detection of staphylococcal genes directly from cerebrospinal and peritoneal fluid samples using a multiplex polymerase chain reaction

A 27-year-old woman receiving electroconvulsive therapy (ECT) for the treatment of depression developed post-ECT headaches refractory to treatment with acetaminophen and various nonsteroidal antiinflammatory drugs. The patient was then treated successfully with oral sumatriptan. This case suggests that sumatriptan may be a useful treatment of post-ECT headaches that are unresponsive to conventional analgesics.     

Genotyping of RHD by multiplex polymerase chain reaction analysis of six RHD-specific exons

BACKGROUND: Qualitative RHD variants are the result of the replacement of RHD exons by their RHCE counterparts or of point mutations in RHD causing amino acid substitutions. For RHD typing, the use of at least two RHD typing polymerase chain reaction (PCR) assays directed at different regions of RHD is advised to prevent discrepancies between phenotyping and genotyping results, but even then discrepancies occur. A multiplex RHD PCR based on amplification of six RHD-specific exons in one reaction mixture is described. STUDY DESIGN AND METHODS: Six RHD-specific primer sets were designed to amplify RHD exons 3, 4, 5, 6, 7, and 9. DNA from 119 donors (87 D+, 14 D- and 18 with known D variants; whites and nonwhites) with known Rh phenotypes was analyzed. RESULTS: All six RHD-specific exons from 85 D+ individuals were amplified, whereas none of the RHD exons from 13 D- individuals were amplified. Multiplex PCR analysis showed that the genotypes of two donors typed as D+ were DIVa and DVa. Red cell typing confirmed these findings. From all D variants tested (DIIIc, DIVa, DIVb, DVa, DVI, DDFR, DDBT) and from RoHar, RHD-specific exons were amplified as expected from the proposed genotypes. CONCLUSION: The multiplex PCR assay is reliable in determining genotypes in people who have the D+ and partial D phenotypes as well as in discovering people with new D variants. Because the multiplex PCR is directed at six regions of RHD, the chance of discrepancies is markedly reduced. The entire analysis can be performed in one reaction mixture, which results in higher speed, higher accuracy, and the need for smaller samples. This technique might be of great value in prenatal RHD genotyping.     

Genotyping by multiplex polymerase chain reaction for detection of endemic hepatitis B virus transmission

A nested polymerase chain reaction (PCR) protocol was developed for rapid genotyping of hepatitis B virus (HBV). During the first PCR round, a universal HBV primer pair was used to amplify the entire pre-S region of the HBV genome. Within the pre-S region, many nucleotide exchanges are observed. These are partly correlated to the serological hepatitis B surface antigen subtypes. Five additional subtype-specific primers were selected from that region which, together with two universal non-group-specific primers, generated specific combinations of two to four DNA fragments of defined sizes. By this approach, 55 hepatitis B surface antigen-positive patients from a pediatric oncology unit in Germany were analyzed. Fifty-four patients who had been infected within 2 years had an identical pattern in the multiplex PCR, suggesting a common source of infection and person-to-person transmission within the unit. One child who was infected 5 years later had a different PCR pattern and, therefore, must have been infected from a different source. Furthermore, 109 serum samples taken from pregnant Cameroonian women and 25 serum samples from their babies taken 6 months after birth were analyzed. In one case, mother-to-infant transmission of the virus was demonstrated. Apart from its role in epidemiological studies on HBV, multiplex PCR may also be a useful tool for rapid genetic analysis in other fields if there is a moderate degree of sequence variation which enables the design of specific primers.     

Identification by polymerase chain reaction fingerprinting of Cryptococcus neoformans serotype AD

Seventy-three Cryptococcus neoformans isolates and eight other yeast strains were studied. Fingerprints produced by priming with (GACA)4 differentiated C. neoformans from all other yeasts tested and identified the five C. neoformans serotypes. Four major bands of molecular size 800, 540, 475 and 410 bp were recognized for serotypes A, AD and D. Two of them were specific for serotype A and the other two for serotype D isolates. Serotype AD strains were identified by five different genotypic patterns in which at least one of the two bands specific for serotype A and D were present in different combinations. On repeated and simultaneously performed genotype and serotype testing of nine strains, the genotypic pattern did not change, whereas serotyping was unstable in three cases. PCR-fingerprinting using (GACA)4 as a primer proved more stable than serology in discriminating among C. neoformans serotypes A, D and AD and was able to distinguish among serotype AD strains.     

The polymerase chain reaction in pathology

Membrane-fixed CD14 acts as a receptor for the protein-bound endotoxin (LPS) complex and mediates the cellular effects of endotoxin. Soluble CD14 (sCD14) is suggested to neutralize circulating LPS, i.e., acting as an endotoxin antagonist. The aim of this study was to elucidate the release of both sCD14 and endotoxin in traumatized patients, starting from the earliest phase after trauma. A total of 15 patients (O ISS = 19, 9-75) suffering major trauma were enrolled in this prospective study. Blood samples were collected as early as immediately at the site of accident, on hospital admission, and thereafter hourly, then daily. For patients (O ISS = 47) died within 24 h because of their severe injuries. Immediately after the accident as well as during the first 2 h after hospital admission, the mean sCD14 levels of surviving patients did not differ from those of healthy volunteers (n = 53). Thereafter, however, sCD14 increased continuously in the trauma group. The concentrations remained elevated throughout the entire observation period. There was, however, no relation between the sCD14 release and the pattern or the severity of injury. In contrast, endotoxin levels revealed a pattern-specific release. The highest plasma concentrations of LPS were observed in patients suffering from (additional) thoracic injury. On the basis of these results we conclude that the release of sCD14 after trauma does not reflect a strict principle such as action/reaction caused by the appearance of endotoxin immediately after the injury. Soluble CD14 is more likely release by an endotoxin-independent mechanism.     

Identification of mycobacteria infecting fish to the species level using polymerase chain reaction and restriction enzyme analysis

An assay is described utilizing PCR technology for a rapid diagnostic test to identify fish infection with Mycobacterium marinum, M. fortuitum and M. chelonae. A 924 bp DNA fragment from a highly conserved area of the mycobacterial 16S rRNA gene was amplified using mycobacteria genus-specific primers and digested with restriction enzymes (BanI and ApaI). This examination yielded unique restriction patterns for each mycobacterial specie enabling identification of mycobacteria infecting fish to the species level. The protocol can be applied to purified DNA, a simple colony preparation or infected fish tissue. This protocol can be completed in 1-2 days.     

Detection of Shigella and enteroinvasive Escherichia coli using polymerase chain reaction

Two oligonucleotide primers were used in a polymerase chain reaction (PCR) procedure to amplify a region of the invasive-associated locus (ial) of Shigella and enteroinvasive Escherichia coli (EIEC). Detection of the amplified product can be done by agarose gel electrophoresis, which is specific and sensitive enough for routine diagnosis of these two pathogens. PCR is done using DNA extracted directly from faeces. The procedure can be completed in 7 h. These findings demonstrate a novel method for rapid, sensitive, specific, and simple diagnosis of diarrhoea caused by Shigella and EIEC.     

Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction

Monitoring genetically modified (GM) bacterial inoculants after field release using conventional culture methods can be difficult. An alternative is the detection of marker genes in DNA extracted directly from soil, using specific oligonucleotide primers with the polymerase chain reaction (PCR). The PCR was used to monitor survival of two GM Rhizobium leguminosarum bv. viciae inoculants after release in the field at Rothamsted. One strain, RSM2004, is marked by insertion of transposon Tn5; the second strain, CT0370, released at the same site, is modified by chromosomal integration of a single copy of the gene from E. coli conferring GUS activity. Both GM strain provide a realistic case study for the development of PCR-based detection techniques. Specific primers were developed to amplify regions of the Tn5 and GUS genetic markers using PCR and conditions optimized for each primer set to routinely detect a signal from 10 fg of purified template DNA, the equivalent of one cell per reaction. Procedures to improve the sensitivity of detection are described, to detect fewer than 50 cells g-1 soil in soil-extracted DNA.     

Detection of human herpesviruses 6 and 7 in heart transplant recipients by a multiplex polymerase chain reaction method

Cystic lesions of the pancreas are relatively uncommon. We describe the case of a young man with a complex cystic mass located within the head of the pancreas. The patient underwent exploration with resection of the mass. Pathology revealed a ciliated epithelial cyst, a rare cystic lesion of the pancreas.     

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.     

Diagnosis of mycotic infections in oncology patients using the polymerase chain reaction]

Diagnosis of mycotic infections is despite the immense effort devoted to this problem still very inaccurate. A new and promising method is the polymerase chain reaction, PCR, which theoretically can detect a single cell. In their original study the authors decided to develop a new method for the detection of fungi by PCR and to compare this examination with post-mortem findings. Thus it was possible to determine sufficiently reliably the sensitivity and specificity of the method. For the detection of fungi the authors selected the sequence coding for a small subunit of ribosomal RNA (18S rDNA). The method is able to detect the amount of DNA from some 10-100 cells. The sensitivity was 90% and the specificity 92%. The method is so far too laborious for common practice, its simplification would be however very useful.     

Detection of avian leukosis virus subgroup J using the polymerase chain reaction

PURPOSE: To compare neutral, external rotation, and abduction external rotation positions of the glenohumeral joint during magnetic resonance (MR) arthrography in the assessment of the joint capsule, biceps-labral complex, and glenohumeral ligaments. MATERIALS AND METHODS: MR imaging with intraarticular administration of gadopentetate dimeglumine was performed in 10 adult cadaveric glenohumeral joints. Fat-suppressed oblique coronal, oblique sagittal, and axial. T1-weighted spin-echo imaging and axial three-dimensional spoiled gradient-recalled imaging were performed with each shoulder in the neutral, external rotation, and abduction external rotation positions. Shoulders were sectioned in the planes that yielded optimal MR images. Anatomic and MR imaging findings were correlated. RESULTS: The biceps-labral complex was best visualized on oblique coronal and axial images obtained in external rotation. Oblique axial abduction external rotation imaging best delineated the inferior glenohumeral ligament but did not improve assessment of the superior and middle glenohumeral ligaments in comparison with findings in neutral and external rotation. CONCLUSION: Although MR arthrography of the glenohumeral joint clearly delineates the biceps-labral complex and glenohumeral ligaments, external rotation of the shoulder optimizes visualization of the former structures. Abduction external rotation is the best position for evaluation of the inferior glenohumeral ligament and anterior capsular attachment.     

High-resolution HLA-DQB1 typing using the polymerase chain reaction and sequence-specific primers

Polymorphism at HLA-DQB1 is known to influence tissue compatibility and disease susceptibility; however, current DQB1 typing methods are unable to distinguish the 32 currently recognized DQB1 alleles. We have developed a 32-reaction PCR-SSP method capable of differentiating all DQB1 alleles that differ in amino acid sequence. This method can resolve all heterozygous combinations of DQB1 alleles, with the exception of several combinations involving alleles not thus far detected in Caucasoid populations.     

Bordetella pertussis diagnosed by polymerase chain reaction

The object of this work was to test the polymerase chain reaction (PCR) for demonstration of Bordetella pertussis (BP) in nasopharyngeal secretions. The method was applied to patients with recently diagnosed pertussis, as verified by BP culture. In order to test the sensitivity and specificity of PCR for the diagnosis of BP, we used known concentrations of BP, Bordetella parapertussis and Bordetella bronchiseptica in aqueous solutions. PCR was furthermore carried out on species of bacteria that might be isolated from the nasopharynx. The applicability of PCR to patient specimens was tested in 25 patients in whose nasopharyngeal secretions BP had been demonstrated after 4-7 days of culture. The detection limit of PCR in aqueous solution was 1-2 BP bacteria per reaction tube. PCR was 100% specific for BP, showing no response with other Bordetella species or other bacteria known to colonize the nasopharynx. Of 25 patient specimens, 16 were PCR-positive 4-7 days after the positive primary culture had been established; only 5 out of 13 patient specimens were positive by repeated conventional nasopharyngeal culture at that time. We conclude that PCR is a possible alternative to culture for the demonstration of BP, as PCR is considerably faster than culture and might be more sensitive.     

Multiplex polymerase chain reaction of Alu polymorphic insertions

Alu sequences are present in humans in excess of 500,000 copies per haploid genome and represent the largest family of short interspersed repetitive elements (SINEs). These mobile genetic elements are ancestrally derived from the 7SL RNA gene and move throughout the genomes of primates by retroposition. Polymorphic Alu insertions have proven to be useful for population studies, paternity determinations and forensic applications. Additionally, a simple polymerase chain reaction (PCR)-based assay has been established to examine these polymorphisms. In the present study, we have applied the technique of multiplex polymerase chain reaction to the Alu polymorphic system. Duplex and triplex PCR reactions were performed for the analysis of five different Alu polymorphic loci in different combinations. This study represents a starting point for further experimentation to improve and eventually optimize Alu multiplex PCR.     

Rapid diagnostic test for hereditary nonpolyposis colon cancer kindred using polymerase chain reaction

PURPOSE: We have identified a mutation in the hMLH1 gene from the proband of a hereditary nonpolyposis colorectal cancer kindred. We wished to develop a rapid test for this specific mutation to facilitate screening of other family members. METHOD: An allele-specific polymerase chain reaction strategy was used to detect a T insertion at the + 3 splice site post exon 9 in the hMLH1 gene. The test was evaluated on DNA in which the mutation status was known. RESULTS: A 130-base pair fragment was reliably amplified using the allele-specific polymerase chain reaction. The test is able to identify the mutant allele and to distinguish between normal, carriers (heterozygous), and tumor DNA samples. The mutant allele is not present in an unrelated hereditary nonpolyposis colorectal cancer cell line or in a sample of the normal population (n=49). CONCLUSIONS: This is a simple, rapid test that can determine carrier status in the members of a kindred at risk for this mutation. This mutation is unlikely to be a polymorphism. This test may now be evaluated in a clinical setting.     

Detection of Coxiella burnetii in cow's milk using the polymerase chain reaction (PCR)

A PCR approach (transposon PCR) with primers based on repetitive transposon-like sequences, which--depending on the isolate--were found at a minimum frequency of 19 on the C. burnetii genome, was established for the highly sensitive and specific detection of C. burnetii. This study describes the analytical detection of C. burnetii in milk, which requires a special preparation method prior to PCR. Because of the low level of C. burnetii particles in milk samples, template DNA was concentrated by a factor of 200, using cetyltrimethylammonium bromide as the precipitation reagent. Using this particular preparation method, even a single C. burnetii particle could be detected in 1 ml milk.