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PubMed2003

Multiplex-PCR and PCR-RFLP assays to monitor water quality against pathogenic bacteria.

In this work we developed and optimized two molecular-based approaches to monitor rapidly, sensitively and specifically bacterial pathogens from three different genera, Escherichia coli, Pseudomonas aeruginosa, and Salmonella spp., directly in waters. To achieve this aim, firstly a multiplex-PCR assay (M-PCR) was optimized using a primer pair specific for each pathogen. Secondly, as a molecular confirmatory test after isolation of the pathogens by classical microbiological methods, PCR-RFLP of their amplified 16S rDNA genes was performed. It was observed from the results that the developed M-PCR assay has significant impact on the ability to detect sensitively, rapidly and specifically the three pathogens directly in water within a short time (5 h from sampling to obtain final results), therefore it represents a considerable advancement over other known more time-consuming and less-sensitive methods for identification and characterization of these kinds of pathogens.

DNA, Bacterial↗

PubMed1997

HPA genotyping by PCR sequence-specific priming (PCR-SSP): a streamlined method for rapid routine investigations.

We describe a streamlined method for the simultaneous identification of alleles of the human platelet antigens (HPA) 1-5. The method employs the polymerase chain reaction with sequence specific primers (PCR-SSP). Although PCR-SSP has been applied to HPA genotyping, all methods previously described have required different reaction mixes and PCR conditions. We have designed a set of sequence-specific primers for HPA 1-5 which react optimally under identical reaction and PCR conditions. Comparative testing with reference samples gave 100% concordance. The advantages of this method include speed; accuracy; smaller sample requirements and no reliance on human typing sera or platelet integrity. The method also has the potential to be applied to amniotic fluid. Simplified DNA techniques will lead to more extensive and proficient platelet antigen typing. This will facilitate accurate laboratory diagnosis of alloimmune thrombocytopenia and the provision of HPA-matched blood products.

Alleles↗

PubMed2002

Specific detection of chikungunya virus using a RT-PCR/nested PCR combination.

Chikungunya (CHIK) virus is enzootic in many countries in Asia and throughout tropical Africa. In Asia the virus is transmitted from primates to humans almost exclusively by Aedes aegypti, while various aedine mosquito species are responsible for human infections in Africa. The clinical picture is characterized by a sudden onset of fever, rash and severe pain in the joints which may persist in a small proportion of cases. Although not listed as a haemorrhagic fever virus, illness caused by CHIK virus can be confused with diseases such as dengue or yellow fever, based on the similarity of the symptoms. Thus, laboratory confirmation of suspected cases is required to launch control measures during an epidemic. CHIK virus diagnosis based on virus isolation is very sensitive, yet requires at least a week in conjunction with virus identification using monovalent sera. We developed a reverse transcription-polymerase chain reaction (RT-PCR) assay which amplifies a 427-bp fragment of the E2 gene. Specificity was confirmed by testing representative strains of all known alphavirus species. To verify further the viral origin of the amplicon and to enhance sensitivity, a nested PCR was performed subsequently. This RT-PCR/nested PCR combination was able to amplify a CHIK virus-specific 172-bp amplicon from a sample containing as few as 10 genome equivalents. This assay was successfully applied to four CHIK virus isolates from Asia and Africa as well as to a vaccine strain developed by USAMRIID. Our method can be completed in less than two working days and may serve as a sensitive alternative in CHIK virus diagnosis.

Alphavirus Infections↗

PubMed2002

Typing of human and bovine Staphylococcus aureus by RAPD-PCR and ribotyping-PCR.

AIMS: To investigate genetic diversity among Staphylococcus aureus and to delineate the geographical distribution of the strains found. METHODS AND RESULTS: RAPD-PCR and ribotyping-PCR were employed for the characterization of Staph. aureus isolates from bovine and nosocomial origin. Among the strains, five to nine groups were distinguished by RAPD-PCR, depending on which primer was used, while ribotyping-PCR distinguished seven ribotypes. CONCLUSIONS, AND SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate the genetic heterogeneity of the strains studied, and the large dissemination of some clones throughout different regions and hosts, findings that may allow the monitoring of Staph. aureus infections.

Animals↗

PubMed2001

Absence of jaagsiekte sheep retrovirus DNA and RNA in bronchioloalveolar and conventional human pulmonary adenocarcinoma by PCR and RT-PCR analysis.

Bronchioloalveolar adenocarcinoma (BAC) morphologically resembles sheep pulmonary adenomatosis (SPA), a contagious ovine pulmonary adenocarcinoma caused by the jaagsiekte sheep retrovirus (JSRV). Previously, positivity for JSRV by immunostaining, reverse-transcription polymerase chain reaction (RT-PCR), and Western blot was reported in most nonmucinous BACs. Our objective in this study was to analyze additional BAC subtypes and conventional adenocarcinomas (CA) to further substantiate this association. Tumor tissue was microdissected from unstained paraffin sections of 26 cases of formalin-fixed, paraffin-embedded BAC (7 mucinous, 17 nonmucinous, 2 sclerosing) and 29 cases of CA. Positive controls consisted of 2 separate paraffin blocks of known SPA. Primer sequences were derived that were capable of hybridizing to all reported strain variants of both the DNA (endogenous) and RNA (exogenous) forms of JSRV. Each sample was tested using both PCR (DNA) and RT-PCR (RNA). All BAC and CA cases were negative for JSRV. Positive controls yielded PCR products that were sequenced and precisely matched the published prototype stain of JSRV. To control for negative effects of tissue fixation, dilutions of positive control tissue were added to BAC and CA samples. Detection of JSRV was evident at 1:50 dilution. Although the possibility of a viral association with BAC cannot be excluded, this study shows that the association with JSRV is probably very weak, if present at all.

Adenocarcinoma, Bronchiolo-Alveolar↗

PubMed2005

Identification of Fritillaria pallidiflora using diagnostic PCR and PCR-RFLP based on nuclear ribosomal DNA internal transcribed spacer sequences.

Fritillaria pallidiflora Schrenk (Liliaceae) is a commonly used antitussive herb. There are 9 species of Fritillaria recorded as herbal drugs in the Chinese Pharmacopoeia. The other species are often marketed as F. pallidiflora, and thus, the therapeutic effects of F. pallidiflora are not achieved. Methods to distinguish F. pallidiflora from the 8 other species of Fritillaria are limited by the current morphological and chemical methods. In this study, we report two molecular authentication methods based on the sequences of nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) regions. For diagnostic PCR, we designed a pair of species-specific primers to authenticate F. pallidiflora. The PCR program consisted of only two steps for every repeated cycle. For PCR-RFLP, we identified a distinctive site which can be recognized by the restriction endonuclease Eco81I in the nrDNA ITS1 region of F. pallidiflora. PCR-RFLP analysis was established to differentiate F. pallidiflora from the other species of Fritillaria. These methods provide effective and accurate identification of F. pallidiflora.

DNA Primers↗

PubMed1990

Reactive site polymorphism in the murine protease inhibitor gene family is delineated using a modification of the PCR reaction (PCR + 1).

Murine protease inhibitor (alpha 1-PI) proteins are encoded by a multigene family which has undergone recent duplication. It has been suggested that the evolution of diversity within this gene family may be driven by unusual selection for novel function at the reactive site of the duplicated members (1,2,3). In an attempt to use polymerase chain reaction (PCR) to generate and sequence clones spanning the polymorphic reactive site region, a PCR artifact was identified and determined to result from heteroduplex formation during the co-amplification of the related sequences in this multigene system. This artifact results in sequences which are combinatorial mosaics of the template sequences. We present a simple and general method (PCR + 1) for overcoming this artifact and demonstrate its application in delineating five distinct alpha 1-PI reactive site sequences in C57BL/6 mice, thus providing sequence information to generate gene-specific probes. The significance of the reactive site diversity in this protease inhibitor gene family is discussed as well as the general applications and limitations of the PCR + 1 technique.

Amino Acid Sequence↗

PubMed1997

Rational primer design greatly improves differential display-PCR (DD-PCR).

Since its conception in 1992, differential display PCR (DD-PCR) has attracted widespread interest. Theoretically an attractive cloning approach, it combines the comparative analysis of several samples with the sensitivity of PCR. Although a large number of studies embracing this technology have been initiated, few novel genes of interest have been identified, suggesting that the method has not realised its potential. The present report shows that by modifying primer design, sampling of differentially expressed genes can be greatly enhanced and relevant genes can be isolated. Using our modified conditions DD-PCR efficiently screens a wide range of gene expression levels, in which differences are represented on a linear scale.

Animals↗

PubMed2005

SiteFinding-PCR: a simple and efficient PCR method for chromosome walking.

In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primer and a vector primer. However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem-loop structure and could not be screened out. This simple method proved to be efficient, reliable, inexpensive and time-saving, and may be suitable for the molecules for which gene-specific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosome walking and obtained 16 positive results from 17 samples.

Arabidopsis↗

PubMed1994

Simple quantitative reverse transcribed-polymerase chain reaction (RT-PCR) method involving recombinant RNA generated by a false-priming PCR product.

A simple and efficient method for preparing rcRNA through competitive RT-PCR has been developed. The basis of this method is the use of a false-priming PCR product including the same primers as a specific product. A 290 bp fragment obtained by two-step PCR was subcloned into a plasmid vector and then the cloned DNA was transcribed into rcRNA. After competitive RT-PCR using sample RNA and rcRNA had been carried out, Southern blot hybridization was performed. The method was applied to determine the amounts of PIMT mRNAs in the rat pituitary. The quantitative analysis indicated that an at least 2-fold difference in PIMT mRNA level can be accurately determined with our method.

Animals↗

PubMed1997

The role of PCR in the diagnosis and management of CMV in solid organ recipients: what is the predictive value for the development of disease and should PCR be used to guide antiviral therapy?

Cytomegalovirus remains a significant source of morbidity and mortality in immunocompromised hosts. The increased sensitivity of molecular diagnostic techniques (PCR, antigenemia) has resulted in our ability to detect viral replication earlier in the posttransplant period, before the onset of symptoms. With the advent of effective antiviral therapy, "preemptive therapy," guided by sensitive, early and specific predictors of CMV disease, has become a realistic objective. Although multiple studies have analyzed the sensitivity and specificity of these tests, their predictive value for the development of disease has not been defined. The purpose of this study was to evaluate the predictive value of a positive CMV PCR in the setting of solid abdominal organ transplantation. A total of 476 PCR assays were performed on 134 transplant recipients (102 kidney, 19 kidney/pancreas, 11 liver, 2 other) either as protocol serial samples or as dictated by clinical events. All samples were concomitantly analyzed using standard virological assays for CMV including culture, shell vial, and serology. Patients with any CMV seropositive donor/recipient (D/R) combination received ganciclovir prophylaxis in conjunction with antilymphocyte induction for 14 days. No subsequent CMV prophylaxis was used. The positive predictive value was 55% in all seropositive donor/recipient combinations. The highest risk group (seronegative recipient of seropositive donor) showed the highest positive predictive value, whereas seropositive recipients of either seropositive or seronegative donors showed positive predictive values of 45% and 25%, respectively. Negative predictive value was 100% for all groups. Early detection of CMV infection has important implications for patient management, including preemptive therapy, which can be guided by PCR, especially in high risk (D+/R-) patients.

Acyclovir↗

PubMed2005

Study of the occurrence of Vibrio vulnificus in oysters in India by polymerase chain reaction (PCR) and heterogeneity among V. vulnificus by randomly amplified polymorphic DNA PCR and gyrB sequence analysis.

The pathogenic bacterium Vibrio vulnificus is widely distributed in estuarine waters throughout the world. In this study, the presence of V. vulnificus in oysters was studied both by conventional culture and DNA-based molecular technique. Following enrichment in alkaline peptone water (APW), the bacteria were lysed and a nested polymerase chain reaction (PCR) for vvhA gene was performed. The effect of duration of enrichment on the sensitivity of detection by PCR was evaluated. The organism was isolated from 43% of samples after 18 h enrichment in APW by conventional culture method. Nested PCR amplifying a fragment of vvhA gene detected the organism in 11%, 60% and 81% of samples following 0, 6 and 18 h of enrichment. All the biochemically identified V. vulnificus strains possessed vvhA gene and belonged to biotype 1. The genetic relatedness among the strains was studied by randomly amplified polymorphic DNA (RAPD) PCR and gyrB sequence analysis. The results suggest the presence of two distinct clonal groups of V. vulnificus in oysters in India. The study demonstrates, for the first time that gyrB sequence analysis could be used to study the genetic diversity of V. vulnificus.

Amino Acid Sequence↗

PubMed2006

Improved template representation in cpn60 polymerase chain reaction (PCR) product libraries generated from complex templates by application of a specific mixture of PCR primers.

Some classes of high G+C content organisms such as the Actinobacteria, which are known through culture-based studies to be present in large numbers in particular microbial communities, are under-represented or even absent from 16S rRNA or cpn60 polymerase chain reaction (PCR) product libraries derived from these templates. Using reference cpn60 sequence data from organisms with high G+C content genomes, a pair of PCR primers were designed which, when used in combination with the previously developed degenerate, universal cpn60 primers, improve the representation of templates with high G+C content. The primers were validated using a combination of traditional and quantitative real-time PCR on both manufactured template mixtures and biological samples. The development and optimization of this specific primer mixture represents an improvement of established methods and a significant advance in the ability to generate cpn60 PCR product libraries that more closely represent the sequence diversity in complex templates.

Bacteria↗

PubMedDate not supplied

Detection of Pneumocystis carinii in oropharyngeal washings by PCR-SHELA and nested PCR.

Oropharyngeal washings (Ophs) from 27 HIV infected patients (18 with P. carinii pneumonia, PCP, and 9 without PCP) were examined for P. carinii using morphological staining and DNA amplification with PCR-SHELA and nested PCR methods. The comparison of these techniques shows that 1. the amplification of P. carinii DNA is more sensitive than (and as specific as) morphological staining; 2. PCR-SHELA is less sensitive than (and as specific as) nested PCR.

AIDS-Related Opportunistic Infections↗

PubMed2003

[Usefulness of arbitrarily primed polymerase chain reaction (AP-PCR) method for DNA fingerprinting analysis of Mycobacterium tuberculosis (MTB); comparison between DNA restriction fragment-length polymorphism (RFLP) method and AP-PCR method].

We have been analyzing cases suspected as outbreak of Mycobacterium tuberculosis in Tokyo using RFLP method. This time we analyzed 27 strains of MTB from 5 cases in two hospitals, a family, member of social activity and stuff of a corporation using both RFLP and AP-PCR methods. At 4 cases, over 80% of strains were same pattern in each cases with RFLP and AP-PCR and were identified as a patient to patients transmission of MTB. At one case, in a hospital, each strains were completely different patterns at both methods, which showed it was not a outbreak case. Results of RFLP and AP-PCR were completely same, which indicates AP-PCR is also useful and rapid method for epidemiological analysis of MTB infection as well as RFLP.

Contact Tracing↗

PubMed2004

Comparison of simultaneous splenic sample PCR with blood sample PCR for diagnosis and treatment of experimental Ehrlichia canis infection.

This report presents evidence that dogs recover from acute canine monocytic ehrlichiosis (CME) after 16 days of doxycycline treatment (10 mg/kg of body weight every 24 h). Blood PCR was as valuable as splenic aspirate PCR for early diagnosis of acute CME. Splenic aspirate PCR was, however, superior to blood PCR for the evaluation of ehrlichial elimination.

Animals↗

PubMed2003

Development of a PCR-enzyme immunoassay oligoprobe detection method for Toxoplasma gondii oocysts, incorporating PCR controls.

Infections caused by Toxoplasma gondii are widely prevalent in animals and humans throughout the world. In the United States, an estimated 23% of adolescents and adults have laboratory evidence of T. gondii infection. T. gondii has been identified as a major opportunistic pathogen in immunocompromised individuals, in whom it can cause life-threatening disease. Water contaminated with feces from domestic cats or other felids may be an important source of human exposure to T. gondii oocysts. Because of the lack of information regarding the prevalence of T. gondii in surface waters, there is a clear need for a rapid, sensitive method to detect T. gondii from water. Currently available animal models and cell culture methods are time-consuming, expensive, and labor-intensive, requiring days or weeks for results to be obtained. Detection of T. gondii nucleic acid by PCR has become the preferred method. We have developed a PCR amplification and detection method for T. gondii oocyst nucleic acid that incorporates the use of hot-start amplification to reduce nonspecific primer annealing, uracil-N-glycosylase to prevent false-positive results due to carryover contamination, an internal standard control to identify false-negative results due to inadequate removal of sample inhibition, and PCR product oligoprobe confirmation using a nonradioactive DNA hybridization immunoassay. This method can provide positive, confirmed results in less than 1 day. Fewer than 50 oocysts can be detected following recovery of oocyst DNA. Development of a T. gondii oocyst PCR detection method will provide a useful technique to estimate the levels of T. gondii oocysts present in surface waters.

Animals↗

PubMed2003

Detection and quantification of Plectosphaerella cucumerina, a potential biological control agent of potato cyst nematodes, by using conventional PCR, real-time PCR, selective media, and baiting.

Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitor P. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence of P. cucumerina in soil, root, and nematode samples. PCR was combined with a bait method to identify P. cucumerina from infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A second P. cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance of P. cucumerina from two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity of P. cucumerina to be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.

Animals↗