Rapid detection of Serpulina hyodysenteriae in diagnostic specimens by PCR.

A PCR assay for the detection of Serpulina hyodysenteriae in diagnostic specimens was developed on the basis of sequence analysis of a recombinant clone designated pRED3C6. Clone pRED3C6, which contained a 2.3-kb DNA fragment unique to S. hyodysenteriae, was identified by screening a plasmid library of S. hyodysenteriae isolate B204 genomic DNA in Escherichia coli by colony immunoblot with the mouse monoclonal antibody 10G6/G10, which was produced against cell-free supernatant antigens from the same isolate. Southern blot analysis of HindIII-digested genomic DNA of S. hyodysenteriae serotypes 1 through 7 and of four weakly beta-hemolytic intestinal spirochetes, including Serpulina innocens, with the 2.3-kb DNA fragment of pRED3C6 indicated that the cloned sequence was present exclusively in the seven serotypes of S. hyodysenteriae. An oligonucleotide primer pair for PCR amplification of a 1.55-kb fragment and an internal oligonucleotide probe were designed and synthesized on the basis of sequence analysis of the 2.3-kb DNA fragment of pRED3C6. Purified genomic DNAs from reference isolates of S. hyodysenteriae serotypes 1 through 9, S. innocens, weakly beta-hemolytic intestinal spirochetes belonging to genotypic groups distinct from those of reference Serpulina spp., other cultivable reference isolates of the order Spirochaetales, and enteric bacteria including Escherichia coli, Salmonella spp., Campylobacter spp., and Bacteroides vulgatus were amplified with the oligonucleotide primer pair in a hot-start PCR. The 1.55-kb products were obtained only in the presence of genomic DNA from each of the nine serotypes of S. hyodysenteriae. The specificity of the 1.55-kb products for S. hyodysenteriae was confirmed on the basis of production of a restriction endonuclease pattern of the PCR products identical to the predicted restriction map analysis of pRED3C6 and positive hybridization signal with the S. hyodysenteriae-specific internal oligonucleotide probe. By using total DNA obtained from normal swine feces inoculated with decreasing concentrations of S. hyodysenteriae cells, the sensitivity of the PCR assay was calculated to be between 1 and 10 organisms per 0.1 g of feces. The PCR assay was 1,000 times more sensitive than conventional culture of dysenteric feces on selective medium. There was complete agreement between the results of PCR assays and anaerobic culture on selective agar medium with diagnostic specimen (n = 9) obtained from six farms on which there were cases with clinical signs suggestive of swine dysentery. Detection of S. hyodysenteriae by PCR amplification of DNA has great potential for rapid identification of S. hyodysenteriae in diagnostic specimens.     

Helicobacter pylori serology using specimens collected on filter paper.

A simple method for the collection of blood specimens on filter paper for serology of Helicobacter pylori was devised. Blood was freshly applied to small Whatman 3 mm filter paper and allowed to dry. Serological activity, as measured by reactivity to H pylori antigens (AMRAD, Australia), was retained after storage for one month at both room temperature and at 4 degrees C. This technique eliminates formal venepuncture, separation of sera, and arduous packaging of specimens to be sent to the laboratory.     

PCR detection of dengue virus using dried whole blood spotted on filter paper

Whole blood dried onto filter paper constitutes a potentially useful material for molecular testing of viruses, including dengue. In order to assess the stability of viral RNA, we carried out dengue-RNA detection in whole blood infected with dengue virus that had been previously spotted onto filter paper. Filter papers were stored at room temperature, 4 and -70 degrees C and processed for PCR assay at intervals of 2, 4, 6 and 9 weeks. Our results demonstrated that dengue-RNA was stable in filter paper for 9 weeks at all tested temperatures. Furthermore, we evaluated these conditions using frozen sera and dried blood samples onto filter paper from 52 patients with confirmed clinical diagnosis of dengue infection. PCR results showed a 100% specificity and 93% sensitivity for dried blood samples. This storage method facilitates the transportation and analysis by nucleic acid amplification techniques even when freezing conditions are not available.     

Direct analysis of filter paper blood specimens for identification of Smith-Lemli-Opitz syndrome using time-of-flight secondary ion mass spectrometry

In this study, time-of-flight secondary ion mass spectrometry was used to distinguish between blood of normal infants and that of individuals with Smith-Lemli-Opitz (SLO) syndrome. SLO syndrome results in an abnormally low concentration of blood cholesterol and an elevated concentration of 7-dehydrocholesterol. Blood was spotted on filter paper and analyzed directly with no extractions or separations. Results showed that using ratios of fragment ions for cholesterol/dehydrocholesterol, patients with SLO and normal individuals could be unambiguously distinguished. Unknown samples from 28 individuals were obtained and identified correctly. Am. J. Med. Genet. 68: 300–304, 1997. © 1997 Wiley-Liss, Inc.     

Novel nested direct PCR technique for malaria diagnosis using filter paper samples

The use of direct nested PCR enables the detection of Plasmodium spp. from blood samples collected on filter papers without requiring the time-consuming procedures associated with DNA extraction. Direct PCR provides a rapid, highly sensitive, and cost-effective alternative to diagnosing malaria using filter paper samples and standard nested PCR.     

Screening for tetrahydrobiopterin deficiencies using dried blood spots on filter paper

Tetrahydrobiopterin (BH4) deficiency among newborns with hyperphenylalaninemia must be rapidly diagnosed and distinguished from classical phenylketonuria (PKU) to initiate immediately specific treatment and to prevent irreversible neurological damage. The characteristic pattern of urinary pterins makes it possible to differentiate between PKU and BH4 deficiencies, and to identify different variants of BH4 deficiency. However, collection, storage, and shipment of urine samples for pterin analysis is cumbersome. A method for the measurement of different pterins (neopterin, biopterin, and pterin) in blood collected on filter paper was developed as a potential alternative to the screening for BH4 deficiencies in urine and for the monitoring of BH4 pharmacokinetics. Pterins pattern in blood spots was comparable with those in plasma and urine. We thus established reference values for pterins in blood spots in patients with hyperphenylalaninemia and identified new patients with GTP cyclohydrolase I deficiency, 6-pyruvoyl-tetrahydropterin synthase deficiency, and dihydropteridine reductase deficiency using dried blood spots on filter paper.     

Rapid identification of mycoplasmas by indirect immunoperoxidase test using small square filter paper.

The indirect immunoperoxidase test using small, square filter paper was used for rapid identification of mycoplasmas. Colonies of type strains of 22 mycoplasma species, 3 acholeplasma species, and three Ureaplasma diversum serogroups were stained by this test with high sensitivity and specificity. All of 49 isolates from bovine materials and cell cultures were easily identified by this test, and the results agreed with those obtained by growth inhibition test. Use of filter paper made it possible to add different kinds of antisera or conjugates to the same agar plate simultaneously and also to save antiserum and conjugate. This test proved to be a simple and useful technique for rapid identification of many mycoplasma species grown on agar medium.     

Analysis of paired serum, urine and filter paper blood specimens for presence of filarial antigen by immunoradiometric assay

Paired serum, urine, and finger-prick whole blood dried on filter paper were analyzed by immunoradiometric assay (IRMA) for filarial antigen using Brugia malayi-specific rabbit antibody. Nine sera and 6 urines from the 10 paired serum-urine samples obtained from individuals with microfilaremia contained IRMA detectable filarial antigen. In contrast, all serum and urine specimens from patients with chronic infections, endemic and non-endemic controls were negative. Whole blood eluted from filter paper spots contained IRMA detectable material; their degree of positivity agreed well with IRMA binding levels obtained with paired urines. Reduced recovery of antigen dried on filter paper was observed at antigen levels less than 10 ng/ml equivalents, presumably due to irreversible absorption onto the filter paper. Urine and finger-prick filter paper blood specimens can be used in the diagnosis of microfilaremic infections that have been associated with circulating antigen in the blood.     

The detection of hemoglobin variants by isoelectrofocusing using EDTA-collected and filter paper-dried cord blood specimens.

A comparative study was conducted aimed at the detection of abnormal hemoglobin conditions (mainly AS, SS, AC, CC, SC, AE, AD, Hb Bart's or gamma 4) by isoelectrofocusing of cord blood samples stored as liquid blood and as dried hemoglobin on filter paper. Analyses were made within four to six days after the collection of the samples; storage conditions mimicked those of testing programs using liquid blood samples (as in Georgia) or dried blood filter paper samples (as in several other states). During analysis of hemoglobin solutions extracted from dried blood samples, considerable difficulties were encountered in detecting significant hemoglobinopathies such as SS and SC, whereas even simple abnormalities such as AS, AC, AD, and AE were also often not diagnosed. Detection of the fast-moving variant Hb Bart's or gamma 4 was not possible. These results again cause doubt regarding the general use of dried blood filter paper samples in newborn hemoglobin testing programs. Perhaps special precautions--such as speed in analyzing the samples, storage at -20 degrees C (or perhaps 4 degrees C) instead of at room temperature, and removal of unstable hemoglobin from the filter paper extract by centrifugation--might eliminate some of the problems that were observed.     

Real-time PCR assay using specimens on filter disks as a template for detection of cytomegalovirus in urine

Since congenital cytomegalovirus (CMV) infection causes late-onset sequelae, the identification of CMV-infected newborns is important. For this purpose, we established a simple real-time PCR assay using a filter disk. Combined with the collection of urine using filter papers placed in the diaper, this assay can make CMV screening more feasible and cost-effective.     

Rapid,efficient method for multiplex amplification from filter paper

Guthrie cards derived from the New York State Newborn Screening Program were utilized to develop a rapid, economical method for amplifying multiple genes to detect mutations that impact public health. These specimens are untraceable to the donor because identifiers are removed and discarded; therefore, these pilot studies were carried out anonymously. The sample preparation requires minimal manipulation, is amenable to automation, and is useful in laboratories which routinely process large numbers of samples, such as those in typical newborn screening laboratories. Multiple gene fragments may be amplified from a 1 mm punch which contains less than 1 μl of whole blood. The blood spots used in these studies contain sufficient material for up to 25 amplification reactions which multiplex at least four different gene fragments each. Since sufficient material remains on the card after the routine testing is complete, this simple assay can greatly expand the efficacy of current newborn screening programs by permitting DNA diagnosis of some disorders when indicated, particularly those in which genotype–phenotype correlations are useful. In addition to newborn screening specimens, this method is also applicable to whole blood from adults after phlebotomy and from lymphoblastoid cell lines. Use of filter paper for DNA analysis is particularly useful for shipped specimens or for population studies whose subjects are refractory to phlebotomy. Hum Mutat 11:404–409, 1998. © 1998 Wiley-Liss, Inc.     

荧光测定法定量测定滤纸片干血斑标本G6PD酶活性的可靠性分析

目的探讨荧光测定法测定滤纸片干血斑标本G6PD酶活性的可靠性。方法随机选取43例门诊孕前咨询者为检测对象,每人采集2ml静脉血抗凝保存同时制备滤纸片干血斑标本。采用G6PD/6PGD比值法测定抗凝血G6PD/6PGD比值,同时采用荧光测定法定量测定滤纸片干血斑标本G6PD酶活性,比较两种方法测定结果。结果荧光测定法检测43份滤纸片干血斑标本,检出1例缺乏（1.57 IU/gHb）;比值法检测43份抗凝血标本,检出1例缺乏（0.76）;两种方法符合率为100%。结论荧光测定法定量测定G6PD酶活性准确性高、简单、快捷、费用低廉,可对滤纸片干血斑标本进行G6PD缺乏症的大规模筛查,适于在G6PD缺乏高发区推广应用。     

Rapid processing of urine specimens by urine screening and the AutoMicrobic system.

A total of 1,500 clean-voided urine specimens were analyzed for the presence of bacteria by urine screening with the Autobac 1 system. The specimens found positive by this method were further processed on the same day for identification and for antimicrobial susceptibility testing on the AutoMicrobic system with the Enterobacteriaceae-plus Card and the General Susceptibility Card, respectively. The inocula for these tests were prepared from the centrifuged and washed growth in the eugonic broth aspirated from the Autobac cuvette chambers. Of 1,500 specimens that were analyzed, 183 contained single isolates of gram-negative bacilli. The results of these rapid procedures were compared with results for the same organisms isolated from urine specimens cultured by the conventional method. The data showed 92.3% agreement for identification and a correlation of 93.6% for antibiotic susceptibility between the two procedures. It is concluded that gram-negative bacilli can be rapidly identified and tested for antimicrobial susceptibility with a high degree of accuracy from the centrifuged eugonic broth after urine screening. These findings also suggest that the AutoMicrobic system provides a rapid and convenient method for same-day processing of positive urine cultures when combined with the urine screening procedure.