Polymerase Chain Reaction In Samples Research Articles

Clinical considerations on Buruli ulcer employing two molecular tests for the detection of Mycobacterium ulcerans in 100 skin biopsies

Buruli ulcer (BU) is an infected cutaneous lesion, the etiological agent of which is Mycobacterium ulcerans. Diagnosis is confirmed by the identification of acid-fast bacilli and culture. In clinically suspicious forms with negative bacteriological or Ziehl-Neelsen (ZN) findings, molecular tests are used. This study compared the concordance of nested polymerase chain reaction (PCR) (targeting IS2404) and PCR (targeting IS2606) in different clinical situations. A total of 57 samples were sourced from 39 BU patients. Control samples (n = 43) were obtained from non-BU ulcers in 38 patients. Samples were divided into two pieces and submitted to, respectively, histological examination and ZN staining, and PCR. Subsamples submitted to PCR were divided and submitted to nested PCR IS2404 and PCR IS2606, respectively. Of the 57 BU biopsies, positive results were obtained by nested PCR in 18 (31.6%) and by IS2606 PCR in 37 (64.9%) cases. Sequencing of the positive samples confirmed the specificity of amplicons in all nested PCR samples and in 26 of 37 (70.2%) samples positive to IS2606. Hence, nested PCR was more specific (100% vs. 93%) and less sensitive (32% vs. 46%) than IS2606 PCR. In the BU samples, nested PCR was negative in 15 instances, and IS2606 PCR was negative in 11 instances in which ZN histology had been positive (false negatives). Both PCRs were positive in six ZN-negative smears. We considered 57 samples from 39 BU patients in various clinical stages and at different times after the beginning of therapy. These provided positive results in 18 cases with IS2404 nested PCR and in 37 cases with PCR IS2606; only 26 of the latter remained positive subsequent to sequencing. Hence, even if IS2404 PCR is considered more specific, in subjects who appear to fail to respond to therapy, it is advisable to also carry out IS2606 PCR. A possible interpretation of the discordance between the two techniques due to unavoidable technical errors as well as to different sensitivity of the two tests at M. ulcerans DNA low concentration (i.e. in recent infection and in well-treated cases) is discussed.

Clinical utility of loop-mediated isothermal amplification assay for the diagnosis of common alpha herpesvirus skin infections

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method with a high specificity, efficiency and speed. No reports exist regarding the usefulness of LAMP for clinically suspected skin infections caused by herpes simplex virus (HSV) or varicella zoster virus (VZV). The aim of this study was to evaluate the clinical usefulness of LAMP in the diagnosis of common cutaneous alpha herpesvirus (HSV type 1 and 2, and VZV) infections. LAMP and real-time polymerase chain reaction (PCR) were performed using swab samples collected from 106 patients with clinically suspected alpha herpesvirus skin infections. The results of LAMP performed with DNA extraction did not differ from those performed without DNA extraction. The sensitivity of LAMP tested against real-time PCR was 96% in herpes simplex, 78% in eczema herpeticum, 93% in herpes zoster and 100% in varicella. No viral DNA was detected by LAMP in all negative real-time PCR samples. Viral DNA load was significantly lower in samples with false-negative LAMP results than in the LAMP-positive samples. LAMP enables confirmation of clinically suspected cutaneous HSV and VZV infections. However, the sensitivity of LAMP is lower than real-time PCR. The accuracy of LAMP may increase if sufficient viral DNA is obtained from lesions. LAMP performed without DNA extraction remains sensitive; thus, LAMP represents a quick and economical method for the diagnosis of common alpha herpesvirus skin infections.

English

Helicobacter pylori is a major etiologic factor in the development of gastritis and peptic ulcer disease. Controversially, the oral cavity has been proposed as a reservoir for this bacterium. The aim of the present study was to determine the prevalence of H. pylori in saliva and dental plaque of adult patients affected by periodontal disease, with or without gastrointestinal symptoms, using polymerase chain reaction (PCR). Suitable PCR primers were designed using sequences of H. pylori DNA from the GenBank database. Chronic periodontal disease was clinically and radiologically diagnosed. The samples for PCR were obtained from periodontal pockets. Cell lysate was centrifuged and used as a DNA source for the PCR assays. The PCR products were analyzed in agarose gels and the sizes of the amplicons were estimated by comparison with DNA size markers. Two samples from each patient were obtained, one from dental plaque and the other from saliva. All salivary samples were negative to PCR isolation of H. pylori, while 13.3% of the plaque samples were positive. All patients with positive PCR isolation of H. pylori had been showing gastrointestinal symptoms. PCR isolation of H. pylori was directly related to the depth of the periodontal pocket. Key words: Periodontitis, gastritis, polymerase chain reaction, Helicobacter pylori.

Molecular determination of Tritrichomonas spp. in aborted bovine foetuses in Eastern Anatolian Region of Turkey

Tritrichomonas foetus is the causative agent of venereal trichomonosis in cattle causing infertility, pyometra and abortions. The objectives of this study were to determine the positivity rate of Tritrichomonas spp. in abomasal content of aborted foetuses from Eastern Anatolian Region of Turkey, using staining, culture and PCR methods and to present the isolates found in the region. A total of 246 abomasal content of aborted foetuses were tested and 14 of 246 (5.7%) were Tritrichomonas spp. positive only by polymerase chain reaction (PCR). Positivity was not attained by staining or culture method. Four of the positive samples in PCR were confirmed to be T. foetus by sequencing of the amplified 5.8S rRNA gene and flanking ITS regions. Nucleotide sequences of TR-Erzurum T. foetus isolates have been entered into the GenBank sequence database under accession numbers KC236423 through KC236426. This preliminary study suggests that future studies are needed on the systematic relationships and epidemiology of T. foetus isolates in the region.

A typical bilateral Toxoplasma retinochoroiditis in a bone marrow transplant patient with negative serum titers

BackgroundToxoplasma retinochoroiditis can have an atypical presentation and be difficult to diagnose in immunocompromised patients. Accurate diagnosis and appropriate treatment is important since the disease can be aggressive in these patients. This paper is a case report with literature review, emphasizing on the diagnosis and treatment of Toxoplasma retinochoroiditis.FindingsA 27-year-old male with chronic myelogenous leukemia with history of bone marrow transplantation presented with floaters in his right eye. Fundus exam showed bilateral, multifocal retinochoroiditis with subsequent development of a mild vitritis. Serum cytomegalovirus and toxoplasmosis antibody titers and syphilis screen were negative. Aqueous polymerase chain reaction (PCR) analysis revealed the presence of Toxoplasma gondii DNA OU. Clindamycin (1.0 mg/0.1 mL) was injected bilateral intravitreal OU twice at 4 days apart with subsequent resolution of retinochoroiditis.ConclusionsWhen evaluating retinochoroiditis in an immunocompromised patient, one must keep a high index of suspicion for atypical presentations of well-known disease entities. Aqueous and vitreous samples for PCR can be useful in obtaining an accurate diagnosis and therefore provide appropriate management for the patient. Intravitreal clindamycin is an option for treatment in these patients.

A direct blood polymerase chain reaction approach for the determination of GP.Mur (Mi.III) and other Hil+ Miltenberger glycophorin variants

GP.Mur (Mi.III) is a glycophorin B-A-B hybrid sialoglycoprotein expressing several potent immunogens, including Mi(a), Mur, and Hil. GP.Mur is considered one of the most important red blood cell (RBC) phenotypes in blood banking in Southeast Asia. However, there are no antibodies commercially available for the screening of GP.Mur RBCs. To develop a direct blood polymerase chain reaction (PCR) approach for the screening of GP.Mur cells, we first confirmed the genomic sequence differences among four GP.Mur and three Mi(a-) samples by sequencing their GYP.Mur and GYPB genes. With these data, we designed PCR primers that best discriminate GYPB and GYP.Mur. Our primer design also allows the detection of other Hil+ glycophorin variants. We also constructed two plasmids--pGBi2i3 and pMiIIIi2i3--which serve as the negative and positive control DNA, respectively, for the PCR procedure. Additionally, we designed a control PCR to be run side by side with the typing PCR. Because of the high specificity of our primers, we found it unnecessary to extract DNA from blood samples for PCR. We have tested this PCR method on 379 fresh and frozen blood samples. The results were further validated by serology and DNA sequencing and were shown to be completely accurate in our hand. We also found that the rapid genotyping method--high-resolution melting--can be a timesaving alternative for DNA sequencing. This direct blood PCR approach for determination of GP.Mur and related Hil+ phenotypes is reliable and economical and is expected to be useful for blood banking in Southeast Asia.

Glass-Based Continuous-Flow PCR Chip With a Portable Control System for DNA Amplification

A glass-based continuous-flow polymerase chain reaction (PCR) chip has been designed and fabricated. The device consists of a glass microfluidic channel, three NiCr heaters, and three Ni thermometers on the silicon substrate. An intelligent temperature-control circuit system has been designed to achieve desirable temperature control (95, 72, and 55 <formula formulatype="inline" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex Notation="TeX">$^{\circ}{\rm C}$</tex></formula> ) at the three temperature zones of the PCR chip. Simulation underneath the microfluidic channel using the finite element method shows that the temperature distribution through the three temperature zones are relatively uniform. A mixture of DNA samples for PCR was allowed to flow through the microfluidic channel under different flow rates. The amplified sample of the target DNA obtained from the PCR chip was then separated by electrophoresis and was analyzed using an ultraviolet analyzer. The result indicates that DNA amplification can be achieved and that its amplification factor depends greatly on the injection rate of the sample. The optimum sample-flow rate is 0.6 <formula formulatype="inline" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex Notation="TeX">$\mu{\rm l}/{\rm min}$</tex></formula> .

Ferrofluid plug as valve and actuator for whole-cell PCR on chip

Ferrofluid plug was first used as valve and actuator to circumvent the evaporation of aqueous fluid in LOC. Moreover, this dynamic plug catered for aqueous fluid expansion during its heating in the microchannel. Whole-cell polymerase chain reaction (PCR) protocol was successfully implemented in a magnetically actuated closed-loop PCR microchip system. Here, the ferrofluid plug was used to reduce the evaporation of the PCR sample in the thermal lysis step. A whole-cell PCR model was used to demonstrate this concept. The DNA plasmid, pUC19, was transformed into DH5α competent cells. Primer pairs were designed to amplify region of interest in pUC19. The transformants were cultured and selected. Whole-cell PCR was performed on intact bacteria to screen for the presence of pUC19, containing the region of interests. Serial dilutions demonstrated the limit of detection for this system to be four bacterial cells. PCR amplifications were verified with regular thermocyclers and results were comparable.

Microbial diagnostics in patients with presumed severe infection in the emergency department

Sepsis in the early stage is a common disease in emergency medicine, and rapid diagnosis is essential. Our aim was to compare pathogen diagnosis using blood cultures (BC) and the multiplex polymerase chain reaction (PCR) test.Methods. At total of 211 patients admitted to the multidisciplinary emergency department of our university hospital between 2006 and 2009 with suspected severe infection from any origin were studied. Blood samples for BC (aerobic and anaerobic) and multiplex PCR were taken for identification of infectious microorganisms immediately after hospital admission. Results of the BC and PCR correlated with procalcitonin concentration (PCT) and clinical diagnosis of sepsis (≥2 positive SIRS criteria) as well as with severity of disease at admission and with clinical outcome measures. Results of the BC were available in 200 patients (94.8%) and PCR were available in 119 patients (56.3%), respectively. In total, 87 BC (43.5%) were positive and identified 94 pathogens. In 45 positive PCRs, 47 pathogens (37.8%) were found. Identical results were obtained in 81.4%. In addition, BC identified 9 Gram-positive and 3 Gram-negative bacteria, while PCR added 5 Gram-negative pathogens. Coagulase-negative staphylococci were detected in blood cultures only (n=20, 21.3%), whereas PCR identified significantly more Gram-negative bacteria than BC. In patients with positive PCR results, the PCT level was significantly higher than in patients with negative PCR (15.0±23.3 vs. 8.8±32.8 ng/ml, p<0.001). This difference was not observed for BC (10.6±25.7 vs. 11.6±44.9 ng/ml, p=0.075). The APACHE II score correlated with PCR (19.2±9.1 vs. 15.8±8.9, p<0.05) and was also higher in positive BC (18.7±8.7 vs. 14.4±8.0, p<0.01). Positive PCR and BC were correlated with negative clinical outcomes (e.g., transfer to ICU, mechanical ventilation, renal replacement therapy, death). In patients admitted with suspected severe infection, a high percentage of positive BC and PCR were observed. Positive findings in the PCR correlate with elevated levels of PCT and high APACHE II scores.

Potential Clinical Usefulness of the Polymerase Chain Reaction Test to Detect Pathogens Causing Sepsis

Objective: A real-time polymerase chain reaction (PCR) test is expected for early and precise detection of pathogens in blood. In this study, we compared the ability of the PCR test and blood culture to detect pathogens in the blood of patients with sepsis. Methods: Patients who were diagnosed as or suspected of having sepsis were included in this prospective observational study. A whole blood sample for PCR test was obtained serially simultaneously with the blood culture sample, and the results were compared. Results: We obtained 93 samples from 26 patients; 69 samples were obtained during the septic condition, and 24 samples were from the non-septic condition. Origins of sepsis were pneumonia in 9 patients, necrotizing fasciitis in 5 patients, and other causes in 12 patients. In the septic condition, rates of positive results were 29.0% for the PCR test and 23.2% for blood culture. Sample contamination occurred in 1 PCR test sample and 5 blood culture samples. In positive PCR samples, 16 of 20 samples were obtained during sepsis after beginning administration of broad- spectrum antibiotics, whereas 5 of 12 samples, except for contaminated samples, were positive in blood culture. Conclusion: In sepsis, the PCR test detected more bacteria than did blood culture even after administration of empirical antibiotics, which might contribute to precise diagnosis of the bacteremic cause of sepsis

Diagnosis of Neonatal Sepsis by Broad-Range 16S Real-Time Polymerase Chain Reaction

Background: The standard diagnostic test (blood culture) for suspected neonatal sepsis has limitations in sensitivity and specificity, and 16S polymerase chain reaction (PCR) has been suggested as a new diagnostic tool for neonatal sepsis. Objectives: To develop and evaluate a new real-time PCR method for detection of bacterial DNA in blood samples collected from infants with suspected neonatal sepsis. Methods: Immediately after blood culture, a study sample of 0.5–1.0 ml whole blood was collected and used for a novel 16S real-time PCR assay. All positive samples were sequenced. Detailed case studies were performed in all cases with conflicting results, to verify if PCR could detect pathogens in culture negative sepsis. Results: 368 samples from 317 infants were included. When compared with blood culture, the assay yielded a sensitivity of 79%, a specificity of 90%, a positive predictive value of 59%, and a negative predictive value of 96%. Seven of the 31 samples with a positive PCR result and a negative blood culture had definite or suspected bacterial sepsis. In five samples, PCR (but not blood culture) could detect a pathogen that was present in a blood culture collected more than 24 h prior to the PCR sample. Conclusions: This study presents an evaluation of a new real-time PCR technique that can detect culture-positive sepsis, and suggests that PCR has the potential to detect bacteria in culture-negative samples even after the initiation of intravenous antibiotics.

One-stop polymerase chain reaction (PCR): An improved PCR method with speedy operation and comparable efficiency

The widely used polymerase chain reaction (PCR) protocol requires several post-cycling steps to visualize amplicons, decelerating PCR sample processing and result calling. “One-stop PCR” was developed by including both the loading buffer and nontoxic staining dye within a single PCR tube, allowing direct loading and simultaneous staining of amplicons for visualization. The efficiency and sensitivity of one-stop PCR were comparable to commercially available PCR kits and it is compatible with downstream uses and is not mutagenic. One-stop PCR robustly minimizes the time and labor required for PCR reaction setup and product visualization. Key words : Polymerase chain reaction (PCR), post-cycling, compatible dye, Gelred.

Multiplex PCR Pathogen Detection in Two Severely Burned Patients With Suspected Septicemia

The case studies reported in this article strive to illustrate the clinical value of multiplex polymerase chain reaction (PCR)-based pathogen detection in patients with burn sepsis. Adult (age ≥18 years) burn patients (≥20% TBSA) presenting with signs and symptoms of burn sepsis were enrolled into a prospective, observational trial. Patients received PCR testing in parallel to routine blood cultures. The authors report two cases in which PCR was used to rapidly detect pathogens in whole blood from burn patients with suspected septicemia. PCR identified Escherichia coli in 5.8 hours in case 1. Blood and sputum cultures required 17 hours for Gram stain results. Empiric ceftriaxone therapy was initiated. Blood cultures required an additional 18 hours to identify the same pathogen detected by PCR. Ceftriaxone was replaced with ciprofloxacin for improved coverage. Follow-up antimicrobial susceptibility results revealed intermediate ciprofloxacin resistance. Meropenam therapy was initiated. In case 2, PCR detected Pseudomonas aeruginosa in 5.45 hours while blood cultures remained negative. Respiratory cultures became positive for P. aeruginosa 2 days later. Serial PCR samples continued detecting P. aeruginosa despite negative blood cultures and appropriate antimicrobial therapy. The patient later became hypotensive and coagulopathic and expired. For both patient cases, PCR identified high-risk pathogens faster than culture methods. In the second patient case, PCR identified the presence of pathogen DNA despite negative cultures before the onset of septic shock and presumptive disseminated intravascular coagulation. These results warrant further investigation to determine the clinical significance of pathogen DNA in burn sepsis.

Designing Polymerase Chain Reaction (PCR) Primer Multiplexes in the Forensic Laboratory

The polymerase chain reaction (PCR) is a common experiment in upper-level undergraduate biochemistry, molecular biology, and forensic laboratory courses as reagents and thermocyclers have become more affordable for institutions. Typically, instructors design PCR primers to amplify the region of interest and the students prepare their samples for PCR and analyze the results. However, primers can also be designed in undergraduate laboratories with students at this level. In a course that focuses on forensic DNA molecular biology for forensic chemistry students, students have used the Applied Biosystems AmpFlSTR SGM Plus kit that amplifies DNA at eleven regions in a single test tube. It is important for forensic chemistry students to be able to design and analyze a single set of primers and, more importantly, create multiplexes of primers. This enables students to more fully understand how the primers and the kits that are routinely employed by the crime laboratories function. Creating a single set of primers does not demonstrate the extent of design and engineering inherent in creating multiplexes or adequately prepare students for research and careers in the field. The in silico method described herein uses free bioinformatics tools and results in student-designed multiplexes for Combined DNA Index System (CODIS) loci. Sample student data are shown.

Microbial diagnostics in patients with presumed severe infection in the emergency department

Sepsis in the early stage is a common disease in emergency medicine, and rapid diagnosis is essential. Our aim was to compare pathogen diagnosis using blood cultures (BC) and the multiplex polymerase chain reaction (PCR) test. At total of 211 patients admitted to the multidisciplinary emergency department of our university hospital between 2006 and 2009 with suspected severe infection from any origin were studied. Blood samples for BC (aerobic and anaerobic) and multiplex PCR were taken for identification of infectious microorganisms immediately after hospital admission. Results of the BC and PCR correlated with procalcitonin concentration (PCT) and clinical diagnosis of sepsis (≥2 positive SIRS criteria) as well as with severity of disease at admission and with clinical outcome measures. Results of the BC were available in 200 patients (94.8%) and PCR were available in 119 patients (56.3%), respectively. In total, 87 BC (43.5%) were positive and identified 94 pathogens. In 45 positive PCRs, 47 pathogens (37.8%) were found. Identical results were obtained in 81.4%. In addition, BC identified 9 Gram-positive and 3 Gram-negative bacteria, while PCR added 5 Gram-negative pathogens. Coagulase-negative staphylococci were detected in blood cultures only (n=20, 21.3%), whereas PCR identified significantly more Gram-negative bacteria than BC. In patients with positive PCR results, the PCT level was significantly higher than in patients with negative PCR (15.0±23.3 vs. 8.8±32.8 ng/ml, p<0.001). This difference was not observed for BC (10.6±25.7 vs. 11.6±44.9 ng/ml, p=0.075). The APACHE II score correlated with PCR (19.2±9.1 vs. 15.8±8.9, p<0.05) and was also higher in positive BC (18.7±8.7 vs. 14.4±8.0, p<0.01). Positive PCR and BC were correlated with negative clinical outcomes (e.g., transfer to ICU, mechanical ventilation, renal replacement therapy, death). In patients admitted with suspected severe infection, a high percentage of positive BC and PCR were observed. Positive findings in the PCR correlate with elevated levels of PCT and high APACHE II scores.

Detection of Mycoplasma pneumoniae in different respiratory specimens

Mycoplasma pneumoniae (M. pneumoniae) is an important community-acquired pneumonia pathogen. Serological test and polymerase chain reaction (PCR) assay are the two main laboratory tests to detect M. pneumoniae now. Little information was compared about the sensitivity and specificity of PCR using different specimens including bronchoalveolar lavage (BAL) and nasopharyngeal aspirate (NPA). The aim of the present study was to evaluate diagnostic values of different specimens by fluorescence quantitative real-time PCR and to find clinical features helpful to diagnose M. pneumoniae pneumonia (MPP). Four hundred and six hospitalized pneumonia children were studied. M. pneumoniae DNA in NPA and BAL samples were detected by fluorescence quantitative real-time PCR. M. pneumoniae-specific IgM was tested by ELISA. MPP were diagnosed based on positive M. pneumoniae-specific IgM in 101 (24.9%) children. The median ages of MPP and non-MPP children were 4.1 and 2.4 years, respectively, with significant difference between them (p < 0.001). Laboratory results including leukocyte count, neutrophil percentage, immunoglobulins, except serum IgM, subgroups of T lymphocyte, and BAL cell count had no significant differences in MPP and non-MPP. BAL macrophage cell percentage was lower in BAL-PCR positive children (p = 0.003), while BAL neutrophil percentage was higher in BAL-PCR positive children (p = 0.007). PCR from NPA and BAL were similar in diagnostic parameters, including sensitivity, specificity, PPV, and NPV (78.6%, 63.4%, 39.8%, and 90.6% for NPA-PCR, respectively; 70.3%, 58.7%, 36.0%, and 85.6% for BAL-PCR, respectively). NPA is better than BAL as PCR sample in MPP diagnosis for similar performance in PCR assay, cheap, and less invasive. BAL is useful in defining local inflammatory condition. Age is the only prefigurative factor in MPP.

Cryptosporidium Pig Genotype II Diagnosed in Pigs From the State of Rio De Janeiro, Brazil

Pigs may represent a source of Cryptosporidium sp. infection to humans. The objective of this study was to identify the Cryptosporidium species present in pigs from the State of Rio de Janeiro, Brazil, and verify what risks pigs represent in the transmission of human cryptosporidiosis, because there is no such information to date in Brazil. Ninety-one samples of pig feces were collected from 10 piggeries in 2 municipalities located in the north and northwest regions of the State of Rio de Janeiro, Brazil. A nested polymerase chain reaction (PCR) protocol to amplify an 830-bp fragment of the small subunit rDNA (SSU rRNA) gene was followed by sequencing of all positive PCR samples. Two samples (2.2%) were Cryptosporidium sp. positive and were identified as pig genotype type II (PGII). This genotype has been observed in an immunocompetent person, in cattle without pigs nearby, and from a potential human source. Its potential for zoonotic transmission is little known and should be rigorously studied.

Nasal Secretion Concentrations of IL-5, IL-6, and IL-10 in Children With and Without Upper Respiratory Tract Viruses

To determine if levels of interleukin (IL) 5, IL-6, and IL-10 or their ratios in nasal secretion are diagnostic of viral upper respiratory tract infections (vURTIs) and coldlike illnesses (CLIs) in children. Longitudinal study of children for vURTIs, CLIs, and concentrations and ratios of nasal cytokines. Outpatient assessments of children. A total of 224 children, aged 1 to 9 years. Concentrations of IL-5, IL-6, and IL-10 in nasal secretions, vURTIs diagnosed by polymerase chain reaction (PCR) detection of upper respiratory tract viruses, and concurrent CLIs diagnosed by parents. Of 1269 secretion samples, 552 (43.5%) were collected during a vURTI (PCR findings positive for an assayed virus [PCR(+)]). A concurrent CLI was diagnosed for 34% of the PCR(+) samples and for 18% of the samples found to be negative by PCR analysis (PCR(-)). Cytokine concentrations and ratios were highly variable and skewed to the lower values. The significance of the cytokine concentrations and ratios as discriminators of groups defined by the presence or absence of virus and of subgroups defined by the presence or absence of a CLI was evaluated using receiver operating characteristic curves. All measures were significant discriminators of the PCR(+) vs PCR(-) groups, and most were significant discriminators of the paired CLI subgroups. The concentration of IL-6 and the IL-5/IL-6 ratio were the best discriminators across all groups and subgroups. However, the sensitivities and specificities of those discriminators at the best cutoff values were on the order of 0.7 for the most extreme pairwise comparison (PCR(+)CLI(+) vs PCR(-)CLI(-)) and lower for the other comparison groups. The low sensitivities and specificities for cytokine-based assignment of specimens to the paired groups and subgroups limit their usefulness for diagnosis of infection or illness.

Presence of a Phytoplasma Associated with Witches’-Broom Disease in Ugni molinae Turcz. and Gaultheria phillyreifolia (Pers.) Sleumer Determined by DAPI, PCR, and DNA Sequencing

Murta (Ugni molinae Turcz.) and common chaura (Gaultheria phillyreifolia (Pers.) Sleumer) are native species of Chile. Plants of both species have shown over-branching like witches’ broom. The causal agents of these symptoms in many plants are phytoplasma. To verify the presence of these microorganisms, DAPI (4’,6-diamidino2-phenylindole) staining analysis and polymerase chain reaction (PCR) were performed in symptomatic and asymptomatic plants. Positive PCR samples were sequenced to identify the pathogens involved. In individuals of both species with witches’ broom symptoms, DAPI staining showed fluorescent bodies in the phloem tissues, but not in asymptomatic plants. Verification by nested-PCR, phytoplasmatic DNA was amplified from diseased murta and chaura, but not in apparently healthy plants. Sequencing of amplified products allowed locating phytoplasma within the ash yellows group (16SrVII) and related to Candidatus phytoplasma fraxini. This is the first report of phytoplasma in Chilean native species. Considering the diversity of plant species infected by the ash yellows group suggests that G. phillyreifolia and U. molinae could be a phytoplasma reservoir for other economically important agricultural crops.

A palmtop PCR system with a disposable polymer chip operated by the thermosiphon effect

A small thermocyling system to perform DNA amplification by polymerase chain reaction (PCR) is presented in this report. PCR reactants are convected along a triangular closed-loop channel in a polymer chip by the thermosiphon effect. In an effort to develop a convection-based PCR for real application, we adopted a molded channel to define the flow path inside the chip, so that the chip may be suitable for disposability together with the merits of LOC; mass production, versatile integration and facile handling. We developed the geometry of the flow loop that made it easier to load the PCR reactants without air pockets inside. Based on systematic simulations and theoretical considerations of buoyant flows, the loop channel was designed to acquire an optimized flow for PCR. A PCR sample was dropped on a chip to fill the loop channel, and the chip was inserted into a slot of a heating block unit that was composed of three metal blocks with different temperatures. The temperature differences within the closed loop gave rise to buoyancy differences and the liquid reactant continuously circulated along the closed loop by the thermosiphon effect. Because there was no loss of time among the temperature shifts in the reaction steps, approximately 10 min were sufficient for the detectable amplification of 127 bp target gene from 10 pg microl(-1) of PCR fragments. In addition, it took 20 min for the mass amplification of 470 bp gene from PCR fragments or genomic DNA. The entire PCR system is compact enough even to be palmtop because it requires only a tiny temperature controller for a self-actuated thermosiphon flow. This thermocycling system would be a prototypical model for the field application of PCR.