Use Of Polymerase Chain Reaction Assay Research Articles

Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering.

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious economic losses worldwide. For example, the citrus tristeza virus killed over 100 million citrus trees globally, while "Candidatus Liberibacter asiaticus" has cost Florida $9 billion. The use of pathogen-tested citrus budwood for tree propagation is key for the management of such pathogens. The Citrus Clonal Protection Program (CCPP) at the University of California, Riverside, uses polymerase chain reaction (PCR) assays to test thousands of samples from citrus budwood source trees every year to protect California's citrus and to provide clean propagation units to the National Clean Plant Network. A severe bottleneck in the high-throughput molecular detection of citrus viruses and viroids is the plant tissue processing step. Proper tissue preparation is critical for the extraction of quality nucleic acids and downstream use in PCR assays. Plant tissue chopping, weighing, freeze-drying, grinding, and centrifugation at low temperatures to avoid nucleic acid degradation is time-intensive and labor-intensive and requires expensive and specialized laboratory equipment. This paper presents the validation of a specialized instrument engineered to rapidly process phloem-rich bark tissues from citrus budwood, named the budwood tissue extractor (BTE). The BTE increases sample throughput by 100% compared to current methods. In addition, it decreases labor and the cost of equipment. In this work, the BTE samples had a DNA yield (80.25 ng/µL) that was comparable with the CCPP's hand-chopping protocol (77.84 ng/µL). This instrument and the rapid plant tissue processing protocol can benefit several citrus diagnostic laboratories and programs in California and become a model system for tissue processing for other woody perennial crops worldwide.

Comparison of a new commercial real-time PCR assay (RealCycler PJIR kit, Progenie Molecular) to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections

The atypical fungus Pneumocystis jirovecii ( P. jirovecii ) is the causative agent of a life threatening pneumonia [ Pneumocystis pneumonia (PCP)] in immunosuppressed patients. PCP diagnosis is essentially based on the detection of the fungus in pulmonary specimens using microscopic examination and polymerase chain reaction (PCR). For the past 20 years, the use of PCR assays has improved the sensitivity of P. jirovecii detection. The main objective of the present study was to compare the efficiency of the RealCycler PJIR kit (Progenie Molecular; pmPCR), to an in-house PCR assay (ihPCR), both assays targeting the mitochondrial large subunit ribosomal rRNA gene. Thirty-four patients were retrospectively enrolled in the study. Fourteen patients were initially diagnosed with PCP, 15 were considered to be pulmonary colonized by the fungus, and five were negative for P. jirovecii detection (negative control group). Archival DNA specimens (extracted from the patient's pulmonary specimens) were examined with ihPCR and pmPCR on the same day using the same real-time PCR instrument (Applied Biosystems 7500 Real-Time PCR system). The ihPCR assay was performed as described elsewhere by Meliani et al. and Totet et al. [1,2] . The pmPCR assay was performed according to the manufacturer's recommendations. Concordant results were obtained with the two PCR assays for all DNA specimens but one (33/34). Thus, the concordance rate between the two methods was evaluated at 97% (Cohen's kappa, 0.905). The discrepant result concerned one specimen from a colonized patient previously known to have a cycle threshold (Ct) value ≥ 35 using ihPCR. This specimen was confirmed to be positive with ihPCR and was negative with pmPCR. Taking into account Ct values for positive samples, the correlation factor between the two PCR methods has been evaluated at 0.97 ( R 2 = 0.9481). Taking into account the Cohen's kappa > 0.8, the RealCycler PJIR kit (Progenie Molecular) appears to be an efficient technique to detect P. jirovecii in patients with PCP and in patients colonized by P. jirovecii .

Species-specific PCR-based assays for identification and detection of Botryosphaeriaceae species causing stem blight on blueberry in China

Botryosphaeriaceae species are important causal agents of blueberry stem blight worldwide. Blueberry stem blight has become an important disease, potentially affecting the quality and production of blueberries in China. It is difficult and time-consuming to identify at the species level using morphological methods. The aim of this study was to develop polymerase chain reaction (PCR) assays for the diagnosis and early detection of latent infections of blueberry stems by Botryosphaeria spp. Species-specific primers, based on the ribosomal DNA internal transcribed spacer region and β-tubulin gene, were designed and selected for use in PCR assays. Three primer pairs, Lt347-F/R for Lasiodiplodia theobromae, Np304-F/R for Neofusicoccum parvum and FaF/Bt2b for Botryosphaeria dothidea, successfully amplified specific PCR fragments of different sizes on pure cultures or from blueberry stems inoculated and naturally infected blueberry plants with three pathogens, respectively. These primers did not amplify any PCR fragments from other blueberry stem disease-associated pathogens, such as Phomopsis spp. and Pestalotiopsis spp. This PCR protocol could detect as low as 100 pg to 1 ng of purified fungal DNA. This PCR-based protocol could be used for the diagnosis and detection of these pathogens from pure cultures or from infected blueberry plants.

Development of PCR Assays for Diagnosis and Detection of the Pathogens Phacidiopycnis washingtonensis and Sphaeropsis pyriputrescens in Apple Fruit.

Speck rot caused by Phacidiopycnis washingtonensis and Sphaeropsis rot caused by Sphaeropsis pyriputrescens are two recently reported postharvest diseases of apple. Infection by these two pathogens occurs in the orchard but remains latent before harvest. Symptoms develop after harvest and are similar to those of gray mold caused by Botrytis cinerea. Accurate diagnosis of these diseases is important during the fruit inspection process, particularly in the instance of fruit destined for export. Early near-harvest detection of latent infections in apple fruit is an important step to implement relevant pre- and postharvest measures for disease control. The aim of this study was to develop polymerase chain reaction (PCR) assays for diagnosis and early detection of latent infections of apple fruit by P. washingtonensis and S. pyriputrescens. Species-specific primers based on the ribosomal DNA internal transcribed spacer region were designed for use in PCR assays. Conventional and real-time PCR assays were developed and validated using fruit inoculated with P. washingtonensis, S. pyriputrescens, or B. cinerea and compared with identifications using traditional isolation-based assays. For wound-inoculated fruit, the PCR assays consistently provided the correct identification of the pathogen used as the inoculant in 6 h of processing time, compared with 5 to 6 days using culture-based methods. Real-time PCR assays effectively detected latent infections in symptomless stem and calyx tissues of fruit that were inoculated with the pathogens in the orchard during the growing season. The PCR assays provide a rapid, accurate method for diagnosis and early detection of these diseases.

Active Surveillance for Methicillin-Resistant Staphylococcus aureus in the Neonatal Intensive Care Unit

We describe our experience using a real-time polymerase chain reaction (PCR) assay for methicillin-resistant Staphylococcus aureus (MRSA) during a period of active surveillance in the neonatal intensive care unit (NICU) from March 2007 until November 2007. To compare PCR with bacterial culture methods and find the screening algorithm that most successfully ensures appropriate isolation of colonized patients. Patients in the NICU were screened for MRSA on admission and weekly thereafter until discharge. Healthcare workers (HCWs) were also screened as part of an outbreak investigation. A total of 599 individuals were screened for MRSA with both a PCR assay and selective bacterial culture. Strain typing was performed on all MRSA isolates to determine clonal relatedness. Twenty-one of 435 infants (4.8%) screened positive for MRSA with the PCR assay. Only 11 patients (52.4%) had concomitant bacterial cultures positive for MRSA. Compared to bacterial culture, the PCR assay had a sensitivity of 100% and a specificity of 97.6%, with a positive predictive value (PPV) of 52.4%. Infants that tested positive for MRSA by both culture and PCR were more likely to have a positive PCR assay result when retested than were those who tested positive by PCR alone (80% vs 20%; P = .02). Strain typing of MRSA isolates identified a common clone in only 2 colonized infants. Our data show that, in our neonatal population, the reproducibility of PCR assay results for culture-negative patients was low compared with the reproducibility of results for culture-positive patients. Furthermore, the low PPV suggests that for nearly half of individuals who were PCR-positive, the result was falsely positive, which argues against the use of PCR assays alone for MRSA screening in the NICU.

Using PCR and culture methods for Mycoplasma testing

Mycoplasma contaminants can be considered important not only for their roles as pathogens but also they may indicate that insufficient care has been taken during vaccine manufacture or quality control. The purpose of this study was to test the poliomyelitis vaccines for Mycoplasma by culture and polymerase chain reaction (PCR) methods. During 2008 to 2009, a total of 47 lots of oral poliomyelitis vaccines were produced by Razi Vaccine and Serum Research Institute (RVSRI) in Iran. They were evaluated by culture and PCR for detection of Mycoplasma. In culture method, PPLO broth and PPLO agar medium were used and in PCR method, universal primers that were selected from the region 16S ribosomal RNA of Mycoplasmas were applied. In culture method, no changes on pH and color in PPLO broth tubes and no colony growth on PPLO agar plates were seen. In PCR method, Mycoplasma DNA was not detected in any of the tested vaccines. It was concluded that the current culture method for Mycoplasmas is reliable to detect viable Mycoplasmas in oral poliomyelitis vaccines, but our results confirmed the use of PCR assay as an efficient supplement to culture method.

Misuse of PCR assay for diagnosis of mollusc protistan infections

Polymerase chain reaction (PCR) assays are useful tools for pathogen surveillance, but they are only proxy indications of pathogen presence in that they detect a DNA sequence. To be useful for detection of actual infections, PCR assays must be thoroughly tested for sensitivity and specificity, and ultimately validated against a technique, typically histology, which allows visualization of the parasite in host tissues. There is growing use of PCR assays for pathogen surveillance, but too often the assumption is made that a positive PCR result verifies an infection in a tested host. This assumption is valid only if the assay has been properly validated for the geographic area and for the hosts examined. Researchers should interpret unvalidated PCR assay results with caution, and editors and reviewers should insist that robust validations support all assertions that PCR results confirm infections.

The usefulness of PCR assay in diagnosing disseminated mycobacterial infection in AIDS patients

Disseminated mycobacterial infections (DMI), caused by Mycobacterium tuberculosis (MTB) and M. avium (MAC), are common complications of late-stage human immunodeficiency virus (HIV) infection [1]. Although the use of highly active antiretroviral therapy (HAART) has substantially reduced DMI incidence [2], disease diagnosis still remains a critical problem for physicians. Both pathologies have non-specific clinical features, unhelpful in the differential diagnosis [3, 4], and microbiological isolation and identification are characterised by long turnaround time and uncertain sensitivity that often oblige clinicians to commence anti-mycobacterial therapy based solely upon clinical suspect. In light of these premises, we evaluated whether the introduction of a molecular method for mycobacterial DNA detection may represent a suitable approach for diagnosis. After optimizing a polymerase chain reaction (PCR) hybridization technique, based on the amplification of a 16rRNA gene sequence using pan-Mycobacterium primers and on the hybridization to two probes, designed to differentiate between M. tuberculosis and M. avium [5], we estimated its reliability and usefulness on blood specimens and/or bone marrow (BM) aspirates. Between March 1999 and April 2004, all of the episodes of suspected DMI investigated with PCR assay performed on blood specimen [6] and a corresponding blood culture (radiometric Bectec AFB system) [7], and/or with a PCR performed on BM aspirate [6] and a corresponding BM analysis (i.e. histopathologic, microscopic and culture exam) [7] were included in the study. Clinical charts were reviewed to assess the diagnosis of disseminated MTB and MAC disease. We drew up two clinical diagnostic criteria: (1) suggestive signs and symptoms of DMI (fever, drenching night sweats, anaemia and weight loss) [3], in addition to blood or BM culture confirmation; (2) suggestive signs and symptoms of DMI in addition to at least two other minor signs or symptoms (diarrhoea, abdominal pain, lymphadenopathy, hepatomegaly, splenomegaly) and/or histopathologic patterns suggestive for mycobacterial infection in patients responding positively to anti-TB or anti-MAC therapy, whose clinical findings excluded other diseases. Patients without exhaustive data to confirm or rule out a DMI were excluded. Data were classified in two-by-two contingency tables to evaluate sensitivity, specificity, negative predictive value and positive predictive value of culture and PCR assay performed on different samples, compared to clinical diagnosis. PCR assay was successfully performed on 117 samples from 95 patients, 71 blood specimens and 46 BM aspirates. Among these, there were 11 paired BM–blood specimens collected from 11 patients available for complete analysis. Eur J Clin Microbiol Infect Dis (2008) 27:163–166 DOI 10.1007/s10096-007-0407-0

Comparison of results for serologic testing and a polymerase chain reaction assay to determine the prevalence of stray dogs in eastern Tennessee seropositive to Ehrlichia canis

To determine the prevalence of stray dogs in eastern Tennessee seropositive to Ehrlichia canis and examine the correlation between results for an ELISA, indirect immunofluorescent antibody (IFA) test, and polymerase chain reaction (PCR) assay. Blood samples obtained from 90 adult dogs admitted to an animal shelter in eastern Tennessee. Serum samples were analyzed for antibodies against E. canis by use of a commercially available ELISA kit, 2 IFA tests, and a PCR assay; testing was performed at the University of Tennessee (TN) and North Carolina State University (NCSU). The PCR amplification was performed by use of DNA extracted from EDTA-anticoagulated blood and primers designed to amplify DNA of Ehrlichia spp. Antibodies against E. canis were detected in only 1 dog by use of the ELISA. By IFA testing at TN, 10 of 90 (11%) dogs were seroreactive against E. canis antigens, all of which had medium to high titers to E. canis. Only 5 of the 10 TN seroreactors were also reactive against E. canis antigens in IFA tests conducted at NCSU, and all 5 had low to medium titers. The DNA of Ehrlichia spp was not amplified in any blood samples by use of PCR assays conducted at the TN or NCSU. The discordant ELISA, IFA, and PCR results obtained in this study were unexpected and may have been related to exposure of dogs to an Ehrlichia species other than E. canis, such as E. ewingii.

Le virus isolé du patient TT (TTV): toujours orphelin deux années après sa découverte

TTV is the acronym for a virus isolated two years ago from a patient whose initials were T.T. It is a naked virus probably belonging to the Circoviridae family. TTV has a particle size of 30-50 nm and possesses a single-strand circular DNA. Epidemiologic data are derived from studies looking for the viral DNA by the polymerase chain reaction (PCR). Important differences between early and recent studies appear to be due to the use of PCR assays based on primers located in different regions of the genome. Based on the most recent studies, the prevalence of TTV infections seems very high in the general population. TTV is present in the feces and would be transmitted through the fecal-oral route. It appears to be a ubiquitous virus, also present in various animal species, from chickens to chimpanzees. No association to any pathology has been identified so far, and TTV infection does not have a significant effect on liver disease.