Universal Polymerase Chain Reaction Research Articles

Simultaneous PCR Detection of Multiple Classes of Integron Integrase Genes for Determining the Presence of Multidrug-Resistant Bacteria in Environmental Samples

Dissemination of multidrug-resistant bacteria, particularly in hospitals, has become a serious public health problem. Integrons impart antibiotic multidrug resistance in gram-negative and some gram-positive bacteria by capturing and then disseminating antibiotic resistance genes. This mechanism plays a major role in contributing to the alarmingly high prevalence of bacterial drug resistance. A universal polymerase chain reaction (PCR) primer set was attempted to design to more sensitively and specifically detect integrons in environmental samples. One set, designated intCiF3(a), intCiF3(b), intCiiiR3(a), and intCiiiR3(b), simultaneously amplifies the conserved region of the tyrosine recombinase gene family between box I and box II. This primer set generates PCR products derived from classes 1, 2, and 3 integron integrases from environmental samples such as wastewater. An unexpected finding of this study was the detection of new putative integron integrase gene sequences. This is the subject of ongoing research, which aims to provide a clear understanding of the risk to human health posed by these genetic elements.

Reliable resequencing of the human dystrophin locus by universal long polymerase chain reaction and massive pyrosequencing

The X-linked dystrophin gene is well known for its involvement in Duchenne/Becker muscular dystrophies and for its exceptional megabase size. This locus at Xp21 is prone to frequent random molecular changes, including large deletions and duplications, but also smaller variations. To cope with such huge sequence analysis requirements in forthcoming diagnostic applications, we employed the power of the parallel 454 GS-FLX pyrosequencer to the dystrophin locus. We enriched the genomic region of interest by the robust amplification of 62 fragments under universal conditions by the long-PCR protocol yielding 244,707 bp of sequence. Pooled PCR products were fragmented and used for library preparation and DNA sequencing. To evaluate the entire procedure we analyzed four male DNA samples for sequence coverage and accuracy in DNA sequence variation and for any potential bias. We identified 562 known variations and 55 additional variants not yet reported, among which we detected a causative Arg1844Stop mutation in one sample. Sanger sequencing confirmed all changes. Unexpectedly, only 3× coverage was sufficient for 99.9993% accuracy. Our results show that long PCR combined to massive pyrosequencing is very reliable for the analysis of the biggest gene of the human genome and open the doors to other demanding applications in molecular diagnostics.

The fatty acid desaturase 3 gene encodes for different FADS3 protein isoforms in mammalian tissues

In 2000, Marquardt et al. (A. Marquardt, H. Stöhr, K. White, and B. H. F. Weber. 2000. cDNA cloning, genomic structure, and chromosomal localization of three members of the human fatty acid desaturase family. Genomics. 66: 176-183.) described the genomic structure of the fatty acid desaturase (FADS) cluster in humans. This cluster includes the FADS1 and FADS2 genes encoding, respectively, for the Delta 5- and Delta 6-desaturases involved in polyunsaturated fatty acid biosynthesis. A third gene, named FADS3, has recently been identified but no functional role has yet been attributed to the putative FADS3 protein. In this study, we investigated the FADS3 occurrence in rat tissues by using two specific polyclonal antibodies directed against the N-terminal and C-terminal ends of rat FADS3. Our results showed three potential protein isoforms of FADS3 (75 kDa, 51 kDa, and 37 kDa) present in a tissue-dependent manner. The occurrence of these FADS3 isoforms did not depend on the mRNA level determined by real-time PCR. In parallel, mouse tissues were also tested and showed the same three FADS3 isoforms but with a different tissue distribution. Finally, we reported the existence of FADS3 in human cells and tissues but different new isoforms were identified. To conclude, we showed in this study that FADS3 does exist under multiple protein isoforms depending on the mammalian tissues. These results will help further investigations to determine the physiological function of FADS3.

Strong Association between Respiratory Viral Infection Early after Hematopoietic Stem Cell Transplantation and the Development of Life-Threatening Acute and Chronic Alloimmune Lung Syndromes

Alloimmune lung syndromes (allo-LS), including idiopathic pneumonia syndrome, bronchiolitis obliterans syndrome, and bronchiolitis obliterans organizing pneumonia, are severe complications after hematopoietic stem cell transplantation (HSCT). In our cohort of 110 pediatric patients, 30 had allo-LS (27.3%), 18 with ideopathic pneumonia syndrome and 12 with bronchiolitis obliterans syndrome. Multivariate analysis showed that respiratory viral infection early after HSCT is an important predictor for the development of allo-LS (P <.0001). This was true for all viruses tested. In multivariate analysis, allo-LS was the only predictor for higher mortality (P = .04). Paradoxically, prolonged administration of immunosuppressive agents because of acute graft-versus-host disease had a protective effect on the development of allo-LS (P = .004). We hypothesize that early infection of the respiratory tract with a common cold virus makes the lungs a target for alloimmunity.

The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay

Real-time polymerase chain reaction (PCR) is the current method of choice for detection and quantification of nucleic acids, especially for molecular diagnostics. Complementarity between primers and template is often crucial for PCR applications, as mismatches can severely reduce priming efficiency. However, little quantitative data on the effect of these mismatches is available. We quantitatively investigated the effects of primer-template mismatches within the 3'-end primer region on real-time PCR using the 5'-nuclease assay. Our results show that single mismatches instigate a broad variety of effects, ranging from minor (<1.5 cycle threshold, eg, A-C, C-A, T-G, G-T) to severe impact (>7.0 cycle threshold, eg, A-A, G-A, A-G, C-C) on PCR amplification. A clear relationship between specific mismatch types, position, and impact was found, which remained consistent for DNA versus RNA amplifications and Taq/Moloney murine leukemia virus versus rTth based amplifications. The overall size of the impact among the various master mixes used differed substantially (up to sevenfold), and for certain master mixes a reverse or forward primer-specific impact was observed, emphasizing the importance of the experimental conditions used. Taken together these data suggest that mismatch impact follows a consistent pattern and enabled us to formulate several guidelines for predicting primer-template mismatch behavior when using specific 5-nuclease assay master mixes. Our study provides novel insight into mismatch behavior and should allow for more optimized development of real-time PCR assays involving primer-template mismatches.

Simultaneous Intraoperative Detection of Methicillin-Resistant Staphylococcus and Pan-Bacterial Infection During Revision Surgery

Periprosthetic infection is one of the most serious complications of arthroplasty, and low-grade infections are particularly difficult to diagnose with use of conventional culture methods. Real-time polymerase chain reaction is a potentially viable way to overcome this detection problem as it is a more rapid and sensitive technique. In the current study, we used intraoperative polymerase chain reaction identification combined with a simple DNA-release method with ultrasonication to diagnose periprosthetic infections during revision surgery. Thirty revision arthroplasty procedures were included in this prospective study. Surgical specimens were obtained intraoperatively, treated with ultrasonication, and then analyzed with real-time polymerase chain reaction. Methicillin-resistant Staphylococcus-specific polymerase chain reaction and 16S rRNA gene universal polymerase chain reaction were performed simultaneously to facilitate both specific and broad-range detection. Specimens obtained from the same sites were also analyzed with microbiologic culture and histopathological evaluation. The specific polymerase chain reaction revealed methicillin-resistant Staphylococcus infection in specimens from six of the thirty operations analyzed in the present study, and the 16S rRNA gene universal polymerase chain reaction analysis was positive for specimens from thirteen operations. Conventional cultures revealed six methicillin-resistant Staphylococcus infections, two Staphylococcus aureus infections, one infection with another Staphylococcus species, and two Streptococcus infections. The sensitivity of the polymerase chain reaction method was 0.87 and the specificity was 0.8 when compared with the combined results of microbiologic culture and histopathological evaluation. The ultrasonication method that we developed for accelerated DNA sample preparation as a replacement for conventional extraction made possible the potential intraoperative identification of periprosthetic infection during revision surgery. The simultaneous detection of methicillin-resistant Staphylococcus and broad-range bacterial infections would be invaluable for the informed selection of antibiotics and also for the formulation of the subsequent treatment strategy (a one-stage or two-stage revision) for the patient.

Microflora associated with successful and failed orthodontic mini‐implants

Mini-implants are used for orthodontic bone anchorage. The reasons for a potential instability or loss of the mini-implants during treatment are multiple. Among other factors, colonization of implants with pathogenic bacteria is discussed. Therefore, the microflora associated with successful and failed mini-implants has been screened. A total of 76 mini-implants collected from 25 patients were observed during regular orthodontic treatment. Bacterial samples of eight failed and - exemplarily - four successful (control) cases were subjected to a universal Bacteria-directed real-time quantitative polymerase chain reaction for quantification in combination with a microarray-based identification of 20 selected species. The failure rate in the present investigation was 10.5%. The bacterial analysis did not reveal any major difference in the total amount or species composition between control and failed mini-implants. However, Actinomyces viscosus was found in four (100%) and Campylobacter gracilis in three (75%) stable controls, whereas both species were rarely found (12.5%) in failed implants. In the present study, the peri-implant sulcus surrounding failed orthodontic mini-implants did not show a specific aggressive bacterial flora.

Large germline deletion of the VHL gene in Chinese families with von Hippel-Lindau syndrome

To investigate the large germline deletion of the VHL gene in Chinese families with von Hippel-Lindau disease (VHL). The large deletion of the VHL gene in 20 unrelated Chinese VHL families was analyzed by using universal primer quantitative fluorescent multiplex polymerase chain reaction (UPQFM-PCR) and GeneScan analysis. Partial and complete VHL gene deletions were detected in 6 probands, including 3 exon 1 deletions, 1 exon 3 and 2 complete deletions. Of the 2 families with the complete deletions, patients developed multi-centric hemangioblastoma in the retina and central nervous system (CNS), and none developed renal cell carcinoma (RCC). Partial and complete VHL gene deletions could be detected in Chinese kindreds with von Hippel-Lindau disease and the test for large deletion of the VHL gene should be implemented in routine DNA diagnosis for VHL disease. Further investigations are required to confirm that entire VHL deletions may be associated with a high risk of hemangioblastomas in the retina and central nervous system.

Uveitis caused by fastidious bacteria

Abstract Purpose The etiologic evaluation of uveitis is frequently unsuccessful if only noninvasive methods are used. We conducted a prospective study to evaluate systematic screening for pathogens of uveitis. Methods All patients with uveitis referred to the participating tertiary ophthalmology departments from January 2001 to September 2007 underwent intraocular and serum specimen collection. The standardized protocol for laboratory investigations included universal polymerase chain reaction (PCR)‐based detection of any bacteria and mycoses, specific PCR‐based detection of fastidious (difficult‐to‐grow) bacteria and herpes viruses, and culture of vitreous fluid. Sera were tested for fastidious bacteria. Results Among the 1321 included patients, infection was diagnosed in 147 (11.1%) patients: 78 (53%) were caused by fastidious bacteria that included spirochetes, Bartonella species, intracellular bacteria (Chlamydia species, Rickettsia species, Coxiella burnetii), and Tropheryma whipplei; 18 by herpes viruses; and 9 by fungi. Bartonella quintana, Coxiella burnetii, Paracoccus yeei, Aspergillus oryzae, and Cryptococcus albidus were found to be associated with uveitis for the first time, to our knowledge. Conclusion We recommend applying a 1‐step diagnostic procedure that incorporates intraocular, specific microbial PCR with serum analyses in tertiary centers to determine the etiology of uveitis.

Universal or broad-range polymerase chain reaction (PCR): a contribution to the detection and identification of bacteria and fungi in clinical practice

The use of techniques for the detection of nucleic acids such as the polymerase chain reaction (PCR) has had a major impact on microbiological analysis, playing an important role in the clinical laboratory. Most of the techniques currently used are designed for specific detection of a particular microorganism. However, infectious agents can also be identified even if genus or species are unknown, using universal primers to amplify bacterial or fungal DNA and then identify the species by sequence (universal or wide spectrum PCR). This methodology is applied in cultures that are difficult to identify using phenotypic techniques, and more recently it is also being used directly in clinical samples, where the detection and identification of the infectious agent by traditional techniques is difficult or not possible.

PCR universal o de amplio espectro: Un aporte a la detección e identificación de bacterias y hongos en la práctica clínica

The use of techniques for the detection of nucleic acids such as the polymerase chain reaction (PCR) has had a major impact on microbiological analysis, playing an important role in the clinical laboratory. Most of the techniques currently used are designed for specific detection of a particular microorganism. However, infectious agents can also be identified even if genus or species are unknown, using universal primers to amplify bacterial or fungal DNA and then identify the species by sequence (universal or wide spectrum PCR). This methodology is applied in cultures that are difficult to identify using phenotypic techniques, and more recently it is also being used directly in clinical samples, where the detection and identification of the infectious agent by traditional techniques is difficult or not possible.

Gab2-Mediated Signaling Promotes Melanoma Metastasis

Metastatic melanoma is a disease with a poor prognosis that currently lacks effective treatments. Critical biological features of metastasis include acquisition of migratory competence, growth factor independence, and invasive potential. In an attempt to identify genes that contribute to melanoma pathogenesis, a genome-wide search using bacterial artificial chromosome array comparative genomic hybridization and single nucleotide polymorphism arrays in a series of 64 metastatic melanoma samples and 20 melanoma cell lines identified increased copy numbers of Gab2 located on 11q14.1. Gab2 is an adaptor protein that potentiates the activation of the Ras-Erk and PI3K-Akt pathways and has recently been implicated in human cancer; however, its role in melanoma has not been explored. In this study, we found that Gab2 was either amplified (approximately 11%) and/or overexpressed (approximately 50%) in melanoma. Gab2 protein expression correlated with clinical melanoma progression, and higher levels of expression were seen in metastatic melanomas compared with primary melanoma and melanocytic nevi. We found that overexpression of Gab2 potentiates, whereas silencing of Gab2 reduces, migration and invasion of melanoma cells. Gab2 mediated the hyperactivation of Akt signaling in the absence of growth factors, whereas inhibition of the PI3K-Akt pathway decreased Gab2-mediated tumor cell migration and invasive potential. Gab2 overexpression resulted in enhanced tumor growth and metastatic potential in vivo. These studies demonstrate a previously undefined role for Gab2 in melanoma tumor progression and metastasis.

Diagnosis of Neonatal Sepsis Using Universal Primer Polymerase Chain Reaction Before and After Starting Antibiotic Drug Therapy

To study universal primer 16S rRNA gene polymerase chain reaction (PCR) for diagnosis of blood culture-positive neonatal sepsis before and after starting antibiotic drug therapy. Prospective study of diagnostic tests. Level III neonatal intensive care unit. Patients Neonates with a fresh episode of clinically suspected sepsis were enrolled; those with major malformations, life expectancy less than 12 hours, or contaminated blood cultures were excluded. Before starting antibiotic drug therapy, PCR (0 hour), blood culture, and sepsis screening (complete blood cell counts, micro-erythrocyte sedimentation rate, and C-reactive protein level) were performed. The PCR was repeated 12, 24, and 48 hours after starting antibiotic drug therapy. The primary outcomes were the sensitivity and specificity of 0-hour PCR for diagnosing blood culture-positive sepsis, and the secondary outcome was the proportion of 0-hour PCR-positive patients who remained positive after antibiotic drug therapy. Of 306 patients evaluated, 242 were included (mean [SD] gestation, 32.2 [3.1] weeks; and mean [SD] birth weight, 1529.2 [597.2] g). Blood culture was positive in 52 patients and 0-hour PCR in 57. The sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios of PCR were 96.2%, 96.3%, 87.7%, 98.8%, 26.1, and 0.04, respectively. Two patients were blood culture positive but 0-hour PCR negative, whereas 7 were 0-hour PCR positive but blood culture negative. Of the 0-hour PCR-positive patients, 7 remained positive at 12 hours and none at 24 and 48 hours after starting antibiotic drug therapy. In 0-hour PCR-positive patients, no predictors of positive 12-hour PCR were identified. Universal primer PCR can accurately diagnose neonatal sepsis before but not after antibiotic drugs are given.

Polymerase Chain Reaction in Intraocular Inflammation

Polymerase chain reaction (PCR) is a technique involving enzymatic amplification of nucleic acid sequences in repeated cycles of denaturation, oligonucleotide annealing and DNA polymerase extension. It is a powerful molecular biologic tool that allows the rapid production of analytic quantities of DNA from small amounts of starting material. PCR can be performed on nearly any ocular specimen or biopsy. For diagnosis of uveitis, the obtained sample is usually an anterior chamber paracentesis or vitreous tap. PCR potentially is more sensitive than culture for detection of many organisms. By utilizing a secondary detection system in concert with the initial PCR reaction, perfect specificity can be assured. The initial application of PCR diagnostics to ophthalmic disease was in the detection of viral uveitis. PCR has also been implicated in studies of noninfectious uveitis. The most common application is HLA typing. A universal bacterial PCR can be very helpful for the diagnosis of bacterial endophthalmitis at an early stage of the disease.

Real‐time PCR of the 16S‐rRNA gene in the diagnosis of neonatal bacteraemia

To evaluate a real-time PCR assay for the diagnosis of neonatal bacteraemia. Two hundred ninety-five plasma samples from 288 newborns with suspected neonatal sepsis were collected prospectively for the purpose of polymerase chain reaction (PCR)-based bacterial detection. A real-time PCR targeting the bacterial gene for 16S-rRNA gene combined with four specific probes designed to detect Gram-negative bacteria, Staphylococcus aureus and coagulase-negative staphylococci (CoNS) was developed. All samples positive in the universal PCR were further sequenced for bacterial identification. When applied to a material from 50 patients with positive blood culture and 245 patients with negative blood culture, the universal PCR showed a sensitivity of 42% (28-57), a specificity of 95% (92-97), a positive predictive value of 64% (45-80), and a negative predictive value of 89% (84-92) (95% confidence intervals in brackets). A new real-time PCR technique was for the first time applied to a well-defined prospectively and consecutively enrolled material of newborns with suspected sepsis, combining the benefits of real-time PCR with specific probes and sequencing. The method managed to detect bacteraemia with high specificity even though the sensitivity was low. Factors causing the low sensitivity are identified and further strategies to develop the method are described.

Activity and DNA contamination of commercial polymerase chain reaction reagents for the universal 16S rDNA real-time polymerase chain reaction detection of bacterial pathogens in blood

Universal 16S rRNA gene polymerase chain reaction (PCR) is a promising means of detecting bacteremia. Among other factors, the PCR reagents play a prominent role for obtaining a high sensitivity of detection. The reagents are ideally optimized with respect to the amplifying activity and absence of contaminating DNA. In this study, it was shown in a universal 16S rDNA real-time PCR assay that commercial PCR reagents can vary greatly among each other in these characters. Only 1 of the 5 reagents tested met the criteria of sensitive detection of pathogen DNA with a minimum of false-positive results. The reagent was validated by the detection of pathogens at low titers using bacterial DNA extracted from blood that was spiked with various Gram-positive and Gram-negative bacteria.

A NOVEL CLINICAL SYNDROME AND DETECTION OF ANAPLASMA OVIS IN MONGOLIAN REINDEER (RANGIFER TARANDUS)

The Tsaatan (or Dhuka) peoples of northern-western Mongolia are one of the few remaining reindeer-herding cultural groups in the world. Recently a disease condition that involves sudden death of reindeer and cases involving fever, lethargy, and pale mucous membranes has been reported. Examination of blood smears collected in the 2005 field season resulted in the identification of intra-erythrocytic inclusions resembling Anaplasma spp. in smears from clinically sick animals. Using universal polymerase chain reaction (PCR) primers for the amplification of the 60 kDa chaperonin gene (cpn60, also known as hsp60 or groEL), we detected sequences corresponding to Anaplasma ovis in reindeer blood samples. Species-specific PCR primers for A. ovis were designed and validated and used to screen blood samples from Mongolian reindeer. Screening of 66 blood samples collected in the 2006 field season resulted in the detection of A. ovis in 80% of the samples. Our results indicate a high prevalence of A. ovis in the Tsaatan reindeer herds and an association with clinical disease that is likely to be anaplasmosis. To our knowledge this is the first report of natural A. ovis infection in reindeer.

A Case of Canine Streptococcal Meningoencephalitis Diagnosed Using Universal Bacterial Polymerase Chain Reaction Assay

A 3-year-old, spayed female, mixed-breed dog was evaluated for acute, progressive neurological disease. Analysis of cerebrospinal fluid (CSF) showed neutrophilic pleocytosis. The dog later developed liver disease, thrombocytopenia, and anemia that were presumably secondary to ceftriaxone administration. Bacterial cultures of blood, urine, and CSF were negative. However, a universal bacterial polymerase chain reaction assay of CSF identified deoxyribonucleic acid from Streptococcus spp. The dog recovered with therapy for streptococcal encephalitis.

Annexin V expression in human placenta is influenced by the carriership of the common haplotype M2

In a group of placentas from patients with preeclampsia or fetal growth restriction, reduced annexin V gene expression in those carrying the M2 haplotype was observed. This is the first "ex vivo" demonstration that annexin V gene expression in placentas is dependent on the M2 haplotype.

High Prevalence of Fastidious Bacteria in 1520 Cases of Uveitis of Unknown Etiology

The etiologic evaluation of uveitis is frequently unsuccessful when noninvasive methods are used. We conducted a prospective study to evaluate systematic screening for pathogens of uveitis. All patients with uveitis referred to the participating tertiary ophthalmology departments from January 2001 to September 2007 underwent intraocular and serum specimen collection. The standardized protocol for laboratory investigations included universal polymerase chain reaction (PCR)-based detection of any bacteria and mycoses, specific PCR-based detection of fastidious (difficult-to-grow) bacteria and herpes viruses, and culture of vitreous fluid. Sera were tested for fastidious bacteria. Among the 1321 included patients (1520 specimens), infection was diagnosed in 147 (11.1%) patients: 78 (53%) were caused by fastidious bacteria that included spirochetes, Bartonella species, intracellular bacteria (Chlamydia species, Rickettsia species, Coxiella burnetii), and Tropheryma whipplei; 18 by herpes viruses; and 9 by fungi. Bartonella quintana, Coxiella burnetii, Paracoccus yeei, Aspergillus oryzae, and Cryptococcus albidus were found to be associated with uveitis for the first time, to our knowledge. We recommend applying a 1-step diagnostic procedure that incorporates intraocular, specific microbial PCR with serum analyses in tertiary centers to determine the etiology of uveitis.