Polymerase chain reaction for detection ofToxoplasma gondii in human biological samples

Using the polymerase chain reaction (PCR), Toxoplasma gondii from gene TGR1E with primers TGR1E-1, TGR1E-2 (standard PCR), and from B1 gene with primers TM1, TM2, TM3 (hemi-nested PCR) was detected in biological samples from 347 individuals (441 biological materials). Of the total of 441 biological materials, T. gondii DNA was detected in 5.2 %; it was positive in the following samples: blood (n = 6), blood from newborns (2), biopsies (2) and samples of progenitor cells (2) (from candidates for bone marrow transplantation). DNA of T. gondii was also revealed in 11 samples (8.3 %) of 120 cases of pregnant women during prenatal examinations. A positive result in the blood was also found in two cases of newborn babies from mothers who were infected in later pregnancy. The positive PCR examination was confirmed by serological methods (ELISA and complement fixation test). Agreement of PCR results and the detection of antibodies against toxoplasma was found in 83.3 %. Rapid PCR examination for the confirmation of acute parasitemia T. gondii is particularly important for the patients in whom the infection may cause serious consequences (e.g., for fetus in pregnant women or for patients suffering from imunosuppression).     

Evaluation of polymerase chain reaction for the detection of Paracoccidioides brasiliensis DNA on serum samples from patients with paracoccidioidomycosis

The aim of this study was to demonstrate the DNA of Paracoccidioides brasiliensis in human serum samples of patients with paracoccidioidomycosis using the polymerase chain reaction (PCR). The diagnosis of paracoccidioidomycosis (PCM) was defined by microscopic observation of the fungus on direct exam or histopathology, culture, and serological positivity. DNA from serum of 33 patients with PCM was extracted and submitted to nested-PCR using primers from the gp 43 gene. Only one sample was positive on nested-PCR. We conclude that the prevalence of fungemia in patients with different clinical forms of PCM is low, limiting the use of serum DNA detection as an alternative diagnostic tool.     

Polymerase chain reaction for the detection of Mycobacterium leprae

A polymerase chain reaction (PCR) using heat-stable Taq polymerase is described for the specific detection of Mycobacterium leprae, the causative agent of leprosy. A set of primers was selected on the basis of the nucleotide sequence of a gene encoding the 36 kDa antigen of M. leprae. With this set of primers in the PCR, M. leprae could be detected specifically with a detection limit approximating one bacterium. This PCR appears to meet the criteria of specificity and sensitivity required for a useful tool in epidemiology and eventually for the control of leprosy.     

Chlamydia-like bacteria in respiratory samples of community-acquired pneumonia patients

Chlamydia-like bacteria, obligate intracellular relatives of Chlamydia trachomatis and Chlamydophila pneumoniae, are widely distributed in nature. Using a two-step nested and semi-nested PCR approach targeting the 16S rRNA gene, we found DNA of Chlamydia-like bacteria in respiratory samples from patients with community-acquired pneumonia. Of 387 cases tested, four (1.03%) tested positive if only sequences showing less than 99.9% 16S rRNA gene sequence similarity to known Chlamydiae were considered. These included for the first time Protochlamydia amoebophila, Waddlia chondrophila, and 'Candidatus Rhabdochlamydia porcellionis'-related sequences. This study extends previous findings suggesting an association of Chlamydia-like bacteria with respiratory disease, but a causal link between these microorganisms and respiratory tract infections has yet to be established.     

Polymerase chain reaction for Mycobacterium tuberculosis complex DNA. Use on negative archival Ziehl-Neelsen cytologic samples

OBJECTIVE: To evaluate the usefulness of a nested polymerase chain reaction (PCR) for Mycobacterium tuberculosis complex on routinely stained cytologic samples from patients with extrapulmonary tuberculosis. STUDY DESIGN: Nested PCR for the detection of a fragment of the IS6110 insertion sequence of M tuberculosis complex was applied to Ziehl-Neelsen-negative archival cytologic slides of serous effusions (pleural [n = 7], peritoneal [n = 1] and pericardial [n = 1]) and a lymph node fine needle aspirate (n = 1) from nine human immunodeficiency virus (HIV)-positive patients with autopsy-proven active extrapulmonary tuberculosis. Malignant effusions and aspirates from nine HIV-positive patients with non-Hodgkin's lymphoma and pleural effusions from seven HIV-negative patients with heart failure were used as controls. DNA was extracted after removing the coverslip and gently scraping the cytologic sample from the slides. RESULTS: In all cases, enough DNA was obtained for PCR without any significant loss of integrity, as demonstrated by PCR positive for HLA-Dq. PCR for M tuberculosis was positive in 8 of the 10 samples (80%) from patients with tuberculosis but also in three samples (30%) from HIV-positive patients in the control group. None of the samples from the HIV-negative patients was positive. CONCLUSION: PCR for M tuberculosis can be reliably performed on archival cytologic slides from extrapulmonary samples, but although it is highly sensitive, it may lead to positive results in immunocompromised patients without any sign of active tubercular disease.     

Polymerase chain reaction as a sensitive and rapid method for specific detection of Mycoplasma pneumoniae in clinical samples

The fast diagnosis of Mycoplasma primary atypical pneumonia is impaired by the lack of routinely available culture methods for isolation of Mycoplasma pneumoniae from clinical specimens. Likewise, serological methods commonly used for diagnosis are insensitive and non-specific. In this study, we have established and applied the polymerase chain reaction (PCR) technique to detect M. pneumoniae DNA in clinical samples originating from the respiratory tract. The PCR results were compared with those from culture and serology tests. To standardize the detection of M. pneumoniae by PCR, we first used DNA from culture grown organisms and clinical samples seeded with M. pneumoniae. PCR amplification was performed with M. pneumoniae-specific primers to amplify 144, 153 and 631 bp DNA fragments by using primer pairs MP5-1/MP5-2, P1-178/P1-331 and P1-178/P1-809, respectively. The amplification of the 631 bp DNA fragment was found to be most sensitive for the detection of M. pneumoniae. Using the most sensitive PCR, a total of 47 respiratory specimens from patients suspected of community acquired pneumonia were tested. While none of the specimens were positive for M. pneumoniae in culture, 6 specimens gave positive results by PCR. In 4 out of the 5 PCR positive samples tested serologically, the results were supported by elevated levels of anti-mycoplasma IgG/IgM/IgA. Thus, these results suggest that PCR is the most sensitive method to detect M. pneumoniae in clinical specimens.     

Polymerase chain reaction for the rapid identification of Clostridium botulinum type A strains and detection in food samples

A polymerase chain reaction (PCR) was developed for the detection of Clostridium botulinum type A, a cause of human botulism. A two primer set and an oligonucleotide detection probe were used to specifically detect Cl. botulinum type A neurotoxin gene (BoNT/A). After 40 cycles of amplification, detection of a 798 bp amplified DNA fragment was carried out by agarose gel electrophoresis and Southern blot hybridization. This assay was able to detect 12.5 fg of purified target DNA or five bacteria per reaction. The sensitivity in artificially contaminated food samples after an 18 h enrichment step ranges from 10 to 10³ bacteria per g according to the type of food samples. No cross-reactions were observed with the other Cl. botulinum toxinotypes and other bacteria found routinely in food. This PCR method may provide a suitable and rapid alternative to standard techniques for detection of Cl. botulinum type A in food samples.     

Polymerase chain reaction assay for the detection of Dendrolimus kikuchii nucleopolyhedrovirus

A polymerase chain reaction (PCR) assay was developed with a pair of primers, designed based on the unique open reading frame 146 of Dendrolimus kikuchii nucleopolyhedrovirus (DekiNPV). DekiNPV DNA served as a template to amplify 426 bp and 655 bp segments. Results from the sequenced PCR products were consistent with the size of selected primer segments. Two primers were applied to assay DekiNPV DNA and detect intact DekiNPV occlusion bodies (OBs), showing a minimum detection of 0.8 fg/mL DNA and 5 OBs/mL, respectively, indicating the existence of DekiNPV in the infected insects. The assay is characterised with high sensitivity, remarkable specificity and early assay, demonstrating its potential for applications in the studies of DekiNPV host range, substitution of DekiNPV hosts, dynamic DekiNPV changes in D. kikuchii population and routine molecular diagnostic procedures.     

Polymerase chain reaction for detection of invasive Shigella flexneri in food.

The polymerase chain reaction (PCR) was used to amplify a 760-base-pair (bp) fragment with the 220-kbp invasive plasmids of enteroinvasive Escherichia coli, Shigella flexneri, Shigella dysenteriae, Shigella boydii, and Shigella sonnei as templates. This PCR product was easily detected by agarose gel electrophoresis. A 210-bp AccI-PstI fragment lying within the amplified region was used as a probe in Southern hybridization blots and showed that the PCR-generated product was derived from the invasive plasmid. The application of PCR as a rapid method to detect enteroinvasive bacteria in foods was tested by inoculating lettuce with 10(4) S. flexneri cells per g in shigella broth base. Plasmid DNA was isolated from cultures of inoculated and uninoculated lettuce in broth after 0, 4, and 24 h of incubation. With the PCR, the 760-bp fragment was generated only from lettuce inoculated with S. flexneri, as shown by gel electrophoresis and confirmed both by Southern blotting and by nucleotide sequencing of the amplified region. Because the isolation of plasmid DNA, the performance of PCR, and gel electrophoresis all can be completed in 6 to 7 h, invasive enteric bacteria can be detected in less than 1 day.     

Polymerase chain reaction detection of nonviable bacterial pathogens.

Polymerase chain reaction (PCR) methodologies for detection of pathogens in environmental samples are currently available. However, positive amplification products for any set of primers only signal that the appropriate target nucleic acid sequences were present in the sample. The presence of the amplification products does not imply that the target organisms were viable. Here we show that PCR will detect nonviable cells, as long as intact target nucleic acid sequences are available. In an environmental water sample, nucleic acids degraded quickly and were not detectable by PCR after 3 weeks even when stored at 4 degrees C. However, these data show that there is a window of opportunity for PCR analyses to result in false positives with respect to viable cells. We further show that care must be taken in the way samples are stored for future PCR amplifications and that filter sterilization of media is not acceptable for long-term preservation of samples for PCR.     

Polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant DNA point mutations

We have microfabricated a flow-through biochip for the analysis of single base mutations in genomic DNA using two different materials: (1) a polycarbonate (PC) chip for performing a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using a universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for sequential PCR/LDR in a continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of gene fragments from genomic DNA; (2) mixing of the resultant PCR product with a LDR mixture via a Y-shaped passive micromixer and (3) ligation of two primers only when the particular mutation was present in the genomic DNA. A PMMA chip was employed as the microarray device, where zip code sequences (24-mer), which were complementary to sequences present on the discriminating primer, were micro-printed into fluidic channels embossed into the PMMA substrate. We successfully demonstrate the ability to detect one mutant DNA in 80 normal sequences with the integrated microfluidic device. The PCR/LDR/hybridization assay using the microchips performed the entire assay at a relatively fast processing speed: 18.7 min for PCR, 8.1 min for LDR, 5 min for hybridization, 10 min for washing and 2.6 min for fluorescence scanning (total processing time=ca. 50 min) with an order of magnitude reduction in reagents compared to bench-top formats.     

Polymerase chain reaction for the detection of Cryptosporidium spp. in cat feces

The objective of this study was to compare a polymerase chain reaction (PCR) assay and a monoclonal antibody-based immunofluorescence assay (IFA) for detection of Cryptosporidium parvum in cat feces. Eight C. parvum-naive DSH cats were orally inoculated with 1 x 10(6) oocysts of a C. parvum human isolate. Fecal samples were collected before inoculation, daily for the next 30 days, and twice weekly until day 85. Methylprednisolone acetate was administered at 20 mg/kg i.m. on days 85, 92, and 99. From days 86 to 115, feces were collected daily and then up to twice weekly until day 126. Immunofluorescence assay was performed after collection of the samples, and then the samples were frozen at -70 C until assayed by PCR. Cryptosporidium parvum was detected by PCR in 101 of 353 samples and by IFA in 52 of 353 samples: 27 samples were PCR positive, IFA positive; 74 samples were PCR positive, IFA negative; 25 samples were PCR negative, IFA positive; and 227 samples were PCR negative, IFA negative. The percentage of concordance between IFA and PCR was 72%. Results of this study suggest that this PCR assay is more sensitive than IFA for detection of C. parvum in cat feces.     

Polymerase chain reaction assay for the detection of Bacillus cereus group cells

Recent investigations have shown that members of the Bacillus cereus group carry genes which have the potential to cause gastrointestinal and somatic diseases. Although most cases of diseases caused by the B. cereus group bacteria are relatively mild, it is desirable to be able to detect members of the B. cereus group in food and in the environment. Using 16S rDNA as target, a PCR assay for the detection of B. cereus group cells has been developed. Primers specific for the 16S rDNA of the B. cereus group bacteria were selected and used in combination with consensus primers for 16S rDNA as internal PCR procedure control. The PCR procedure was optimized with respect to annealing temperature. When DNA from the B. cereus group bacteria was present, the PCR assay yielded a B. cereus specific fragment, while when non-B. cereus prokaryotic DNA was present, the consensus 16S rDNA primers directed synthesis of the PCR products. The PCR analyses with DNA from a number of non-B. cereus confirmed the specificity of the PCR assay.     

Comparison of polymerase chain reaction and conventional culture for the detection of legionellae in cooling tower waters in Singapore

A total of 80 cooling tower water samples were investigated for legionellae using both cultural and polymerase chain reaction (PCR) methods. PCR was performed with the Perkin Elmer EnviroAmp^™ Legionella kit. Forty-seven samples (58·8%) were found positive by both methods ; 29 samples (36·3%) were positive by PCR only, while four samples (5%) showed PCR inhibition despite the adoption of the more stringent sample preparation protocol especially designed to eliminate inhibitors.     

New microbiota found in sputum from patients with community-acquired pneumonia

Community-acquired pneumonia （CAP） is a major concern in hospitals and the bacterial community of which has not been systemically discussed yet. Sputum from patients in the acute stages is a kind of accessible sample reflecting its fea- tures. In our study, we analyzed 45 sputum samples from 45 patients with CAP. Eighteen sputum samples from healthy people were chosen as the controls. Pyrosequencing of the 16s rDNA V3 hypervariable regions of aH the bacteria con- tained in the sputum was used as a culture-independent method to disclose the community constitution. Also, our published data for hospital-acquired pneumonia （HAP） in sputum was used for comparison. By pyrosequencing, 〉90,000 DNA reads were detected. After being analyzed by tools in the Ribosomal Database Project, the reads were clas- sified into five main phyla and 〉100 genera. At the phyla level, the reads＇ distribution of CAP is similar to that of healthy people and at genera level, the occurrence of each genus possesses their feature in three categories. Genera such as Streptococcus and Neisseria showed stability in their percentages, indicating that such genera are rarely affected by exogenous bacteria or antibiotics. The role of other genera such as Moraxella and Rothia in CAP should be emphasized. According to our analysis, the bacterial communities of CAP are with slight change when compared with those of healthy people, but have a large gap between HAP. Meanwhile, Rothia might be an important endogenous pneumonia-causing factor.     

应用PCR方法检测部分血液制品中HCV-RNA

应用市售丙型肝炎ＰＣＲ检测试剂盒,对１９９４ １９９６年用不同病毒灭活工艺生产的人凝血因子类制品、人静注丙球和人血白蛋白进行ＨＣＶ ＲＮＡ检测。结果表明,检测六种血液制品共２１批,１批阳性,占４８％。     

Polymerase chain reaction-gene probe detection of microorganisms by using filter-concentrated samples.

To detect low levels of microorganisms in environmental samples by using polymerase chain reaction (PCR)-gene probe detection, samples were concentrated by filtration. Fluoropore (Millipore Corp.) filters were compatible with PCR DNA amplification, whereas various other filters including nitrocellulose and cellulose acetate filters inhibited PCR amplification. By concentrating cells on Fluoropore filters and releasing the DNA by freeze-thaw cycling, PCR DNA amplification could be performed without removing the filter. Concentration with Fluoropore FHLP and FGLP filters permitted the detection of single cells of microorganisms in 100-ml samples by PCR-gene probes.     

Polymerase chain reaction detection of potentially pathogenic free-living amoebae in dental units

Several genera of amoebae can be found in water from dental units and on the inner surface of waterlines. The presence of bacterial biofilms on these surfaces is thought to favor the proliferation of amoebae. Potentially pathogenic Acanthamoeba and Naegleria spp. may be an infection risk for patients through contact with open surgical sites or aerosolization. A polymerase chain reaction of DNA extracted from pelleted samples showed that Acanthamoeba spp. and Naegleria spp. were present in water from dental units, suction lines, and suction filters at the dental clinic of the Université de Montréal. Acanthamoeba spp. were detected in 24.2% of 66 samples and Naegleria spp. in 3.0%. We discuss the infection risk associated with these results.