Conventional PCR Techniques Research Articles

PCR‐based techniques to determine diet of the Australian sea lion (Neophoca cinerea): a comparison with morphological analysis

AbstractComprehensive dietary information for the endangered Australian sea lion (Neophoca cinerea) is currently limited by the deficiency and poor quality of identifiable prey remains recovered from regurgitate and faeces and the difficulty of observing feeding in the wild. In this study, we investigated DNA‐based prey detection methods using conventional (end‐point) and quantitative real‐time PCR (qPCR) on faeces collected from two captive Australian sea lions fed experimental diets of whole teleost fish, squid and shark tissue. PCR prey detection methods using the mitochondrial cytochrome oxidase subunit I (COI) and 16S genes combined with clone sequencing were compared with prey identified using traditional hard part analysis. The molecular results indicated that prey DNA was degraded. However, prey amplification was successful by targeting short (71 bp) DNA fragments. Both conventional PCR and qPCR techniques significantly increased prey detection compared with analysis of hard parts. For both sea lions, the hard part analysis was constrained by sporadic and extremely low recovery of fish otoliths (<2%), and cephalopod beaks were not recovered from the 116 squid fed. Comparisons between PCR techniques indicated comparable prey detection frequencies for all species tested; however, the sensitivity and greater resolution of qPCR improved prey detection by ~25% in one sea lion fed the experimental squid and perch. The detection of squid DNA ≤ 6 day post‐ingestion by qPCR further exhibits the ability and potential of this method to detect low concentrations of infrequent or pulse prey. This study highlights the use of DNA‐based analysis to detect prey taxa in the absence of identifiable hard prey remains.

Quantitative PCR in epidemiology for early detection of visceral leishmaniasis cases in India.

IntroductionStudies employing serological, DTH or conventional PCR techniques suggest a vast proportion of Leishmania infected individuals living in regions endemic for Visceral Leishmaniasis (VL) remain asymptomatic. This study was designed to assess whether quantitative PCR (qPCR) can be used for detection of asymptomatic or early Leishmania donovani infection and as a predictor of progression to symptomatic disease.MethodsThe study included 1469 healthy individuals living in endemic region (EHC) including both serology-positive and -negative subjects. TaqMan based qPCR assay was done on peripheral blood of each subject using kDNA specific primers and probes.ResultsA large proportion of EHC 511/1469 (34.78%) showed qPCR positivity and 56 (3.81% of 1469 subjects) had more than 1 calculated parasite genome/ml of blood. However, the number of individuals with parasite load above 5 genomes/ml was only 20 (1.36% of 1469). There was poor agreement between serological testing and qPCR (k = 0.1303), and 42.89% and 31.83% EHC were qPCR positive in seropositive and seronegative groups, respectively. Ten subjects had developed to symptomatic VL after 12 month of their follow up examination, of which eight were initially positive according to qPCR and among these, five had high parasite load.DiscussionThus, qPCR can help us to detect significant early parasitaemia, thereby assisting us in recognition of potential progressors to clinical disease. This test could facilitate early intervention, decreased morbidity and mortality, and possibly interruption of disease transmission.

Whole-genome analysis of herbicide-tolerant mutant rice generated by Agrobacterium-mediated gene targeting.

Gene targeting (GT) is a technique used to modify endogenous genes in target genomes precisely via homologous recombination (HR). Although GT plants are produced using genetic transformation techniques, if the difference between the endogenous and the modified gene is limited to point mutations, GT crops can be considered equivalent to non-genetically modified mutant crops generated by conventional mutagenesis techniques. However, it is difficult to guarantee the non-incorporation of DNA fragments from Agrobacterium in GT plants created by Agrobacterium-mediated GT despite screening with conventional Southern blot and/or PCR techniques. Here, we report a comprehensive analysis of herbicide-tolerant rice plants generated by inducing point mutations in the rice ALS gene via Agrobacterium-mediated GT. We performed genome comparative genomic hybridization (CGH) array analysis and whole-genome sequencing to evaluate the molecular composition of GT rice plants. Thus far, no integration of Agrobacterium-derived DNA fragments has been detected in GT rice plants. However, >1,000 single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) were found in GT plants. Among these mutations, 20–100 variants might have some effect on expression levels and/or protein function. Information about additive mutations should be useful in clearing out unwanted mutations by backcrossing.

Abstract 4674: Finding cancer biomarkers in platelets

Abstract Introduction: In the development of personalized medicine we need a window in to the tumor; it should be minimal invasive, being able to find the disease and follow how the tumor alters its behaviour to circumvent the treatment. Platelets could be one such window, with its ability to sequester tumor-derived material during its passage through the circulation. Material & Methods: With conventional quantitative PCR and microarray techniques “potential” biomarkers were analysed from WTA (whole transcriptome amplified) platelet-derived RNA isolated from cancer patients and healthy controls. Results: We demonstrate that platelets contain several cancer-associated RNA biomarkers. In addition, there was a distinct biomarker signature in platelets from cancer patients of different grades as compared to normal control subjects. Conclusion: These findings are preliminary, and need to be validated in a larger patient cohort. However, they support our earlier findings in gliomas that platelets may carry the needle from the haystack that will give us a window into the cancer biology of individual patients. Citation Format: Lee-Ann Tjon-Kon-Fat, Marie Lundholm, Thomas Wurdinger, Camilla Thellenberg, Anders Widmark, Pernilla Wikström, Jonas Nilsson. Finding cancer biomarkers in platelets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4674. doi:10.1158/1538-7445.AM2014-4674

An improved UPLC method for the detection of undeclared horse meat addition by using myoglobin as molecular marker

In 2013, following the scandal of the presence of undeclared horse meat in various processed beef products across the Europe, several researches have been undertaken for the safety of consumer health. In this framework, an improved UPLC separation method has been developed to detect the presence of horse myoglobin in raw meat samples. The separation of both horse and beef myoglobins was achieved in only seven minutes. The methodology was improved by preparing mixtures with different composition percentages of horse and beef meat. By using myoglobin as marker, low amounts (0.50mg/0.50g, w/w; ∼0.1%) of horse meat can be detected and quantified in minced raw meat samples with high reproducibility and sensitivity, thus offering a valid alternative to conventional PCR techniques.

Real time PCR in childhood tuberculosis: a valuable diagnostic tool.

The present study was conducted to detect and quantitate Mycobacterium tuberculosis from various body fluid specimens of cases of tuberculosis by real time PCR technique and compare results with conventional PCR technique and culture. One hundred fifteen children (<18 y) with tuberculosis (diagnosed as per IAP guidelines) and 32 disease matched controls from the Department of Pediatrics, S.N. Medical College, Agra, were included in the study. Different body fluids (CSF, gastric aspirate, pleural fluid, ascitic fluid and lymph node aspirate) were subjected to culture, conventional PCR targeting insertion sequence 1S6110 and Real time PCR targeting 16srRNA of Mycobacterium tuberculosis. Real time PCR showed significantly better results than culture in all body fluids (p < 0.05). It was superior to conventional PCR in CSF (p < 0.05) but showed comparable results in gastric aspirate, pleural fluid, ascitic fluid and lymph node aspirate (p > 0.05). Hence, real time PCR is a promising diagnostic tool for childhood tuberculosis, particularly tubercular meningitis.

Investigation of sensitivity, specificity and accuracy of Tetra primer ARMS PCR method in comparison with conventional ARMS PCR, based on sequencing technique outcomes in IVS-II-I genotyping of beta thalassemia patients

PurposeBeta thalassemia is one of the most important hematic diseases all around the world and solving the problems caused by this abnormality is strongly dependent on precise detection and reliable screening of high-risk couples. The aim of our study was the investigation of sensitivity, specificity and accuracy of Tetra primer ARMS PCR method comparing with conventional ARMS PCR, based on sequencing technique outcomes for genotyping of IVS-II-I mutation in beta thalassemia patients. MethodsFifty seven samples including two homozygote, 49 heterozygote and 6 normal specimens were analyzed by Tetra primer ARMS PCR and conventional ARMS PCR methods. DNA was extracted by the standard method of salting out for leukocyte genomic DNA extraction of blood specimens and a high pure PCR template preparation kit was used for DNA purification of CVS samples. The results obtained by Tetra primer ARMS PCR and conventional ARMS PCR methods were compared with gold standard technique, i.e. sequencing. ResultsAll three parameters including specificity, sensitivity and accuracy were 100% for Tetra primer ARMS PCR method, while they were 100%, 92.45% and 92.7% for conventional ARMS PCR technique respectively. Comparing with Tetra primer ARMS PCR which represented 100% agreement with sequencing method, conventional ARMS PCR technique only showed 47.1% agreement, because of 4 discordant results. ConclusionTetra primer ARMS PCR method is an almost reliable, sensitive and accurate technique and it is suggested that it can be used as a complementary method for diagnostic cases instead of conventional ARMS PCR method. This suggestion originated with perfect rate of agreement between outcomes of sequencing method, as a gold standard method of detecting the mutations, and Tetra primer ARMS PCR technique comparing with conventional ARMS PCR method.

Pulmonary nocardiosis associated with cerebral abscess successfully treated by co-trimoxazole: a case report

Nocardiosis is an acute or chronic infectious disease caused by the soil-borne filamentous bacteria belonging to the genus Nocardia. The organisms opportunistically infect both immunocompromised and immunocompetent individuals. The lungs are the primary site of infection and brain abscess is, by far, the most common complication following nocardial metastasis from pulmonary lesions. Although surgical intervention must always be considered in the treatment of nocardial brain abscess, it can obviously be cured by antibiotic therapy alone. This report describes a case infected by Nocardia cyriacigeorgica. Identification of the infectious agent was achieved by conventional and semi-nested PCR techniques. A 55-year-old woman with fever was referred to the infect disclinic of Imam Khomeini hospital in Tehran and was hospitalized after clinical assessment. She was a kidney transplant recipient for 4 years and was taking immunosuppressive treatment including azathioprine and methylprednisolone. Follow-up of the patient by CT scan revealed pulmonary infection and cerebral lesions. Specimens of the brain lesions contained filamentous bacteria. The patient received a combination of co-trimoxazole and ceftriaxone and brain abscesses as well as lung inflammation disappeared gradually during the course of antibiotic therapy within 3 months. The patient was discharged from the hospital after 2 months of therapy.

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One hundred (100) staphylococci were isolated from several Egyptian hospitals and laboratories which include 85 clinical isolates and 15 from hospital surroundings. The isolates were identified by conventional and molecular techniques. Fifty (50) isolates were identified as Staphylococcus aureus (SA), 40 were Staphylococcus epidermidis (SE) while 10 were identified as Staphylococcus species (SS). Upon testing the resistance to methicillin and vancomycin, it was found that resistance is dominant in isolates from clinical samples than those from the surrounding surfaces. Moreover, the resistance to methicillin was higher than that to vancomycin. Multiplex polymerase chain reaction was carried out to characterize the staphylococci-specific region of 16S rRNA gene, mecA gene associated with methicillin resistance and the virulence marker-associated genes Panton-Valentine leukocidin (PVL) lukS/F-PV genes which are responsible for leukocyte destruction and tissue necrosis. All the methicillin resistant staphylococci (MRS) were found to be mecA+ while only five MRS carried lukS/F-PV genes. On the other hand, 30% of the methicillin sensitive staphylococci (MSS) were found to harbor the mecA gene while lacking the PVL. The results highlight the important role of horizontal gene transfer of virulence genes between staphylococci. In addition, this study indicates that the use of multiplex PCR is not sufficient for antibiotic susceptibility prediction and thus the simultaneous use of conventional and multiplex PCR technique is required for the identification of staphylococci and determination of their antibiotic susceptibility. Key words: Methicillin resistance, Staphylococcus aureus, Staphylococcus epidermidis, mecA, Panton-Valentine leukocidin (PVL).

Detection of transgenic events in maize using immunochromatographic strip test and conventional PCR

With the growth in the transgenic market, fast and economically viable methodologies are necessary for undertaking transgene detection tests, both for identification of contamination in seeds and in grain. Seeds from commercial conventional GNZ 2004, and transgenic VT-Pro (MON89034), Roundup Ready (NK603) and Herculex (TC1507) maize cultivars were used. In order to simulate different levels of contamination, the transgenic seeds were mixed with conventional seeds at levels of 0.2%, 0.4%, 1.0% and 1.6% for VT-Pro, and 0.2%, 0.5%, 0.8% and 1.2% for Roundup Ready and Herculex. The lateral flow membrane strip test was performed in the whole seed, endosperm and embryo. For evaluation of the specificity of the technique in detection of the TC1507 event by means of the conventional PCR technique, seeds of the commercial maize hybrid GNZ 2004 were used as negative control, and the maize hybrid 2B655Hx as positive control. In order to simulate different levels of contamination, transgenic seeds were mixed with conventional seeds at the levels of 10%, 5%, 1%, 0.5% and 0.1%. Seeds from each sample were crushed, and then DNA extraction was performed by the CTAB 2% method. Using the immunochromatographic strip, it was possible to evaluate the expression of proteins related to the VT-Pro, Roundup Ready and Herculex events when whole seeds were used at the 0.2% level of contamination, whereas by the conventional PCR technique, it was possible to detect the TC1507 event in samples with 1% contamination.

Isolation and Molecular Characterization of Mycoplasma gallisepticum and Mycoplasma synoviae in Chickens in Sudan.

The current study described the isolation and molecular detection of Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae from tracheal swabs of diseased birds showing signs of respiratory distress in selected commercial (layer and broiler) farms and from yolk and an open air of pens of vaccinated breeder flocks in Sudan. A number of 45 Mycoplasma isolates were recovered from chickens in Khartoum, Gezira, and Equatoria states in Sudan. Of these, eight Mg and three Ms isolates were identified using growth inhibition and rapid serum agglutination (RSA) tests. The conventional PCR technique was applied to amplify 140 bp and 720 bp DNA fragments for the Mg and Ms, respectively. This research confirmed vertical and horizontal transmission of Mg from breeder farms through detection of Mg in yolk of fertile eggs and an air of pens despite previous vaccination. PCR is considered a rapid, sensitive, and cheap method and it will improve the diagnosis of Mycoplasma in chickens.

Dry-reagent-based PCR as a novel tool for the rapid detection of Clostridium spp.

Improved conventional PCR techniques are required for the rapid on-site detection of human and animal diseases. In this context, a PCR method using dry-stored reagents intended for the detection of Clostridium spp. is presented. Basic PCR reagents (BSA, PCR buffer, MgCl₂ and primers), which were dried on polyolefin matrices, showed stability at ambient temperatures for up to 10 months without any loss of functionality. An outstanding advantage of our amelioration is the elimination of PCR process errors caused by the improper storage and handling of liquid reagents. Moreover, our PCR-based amplification can be performed in less than 30 min, saving time compared with conventional detection methods. Thus, dry-reagent-based PCR is implementable in a suitcase-like modular device for the rapid on-site detection of microbial pathogens such as blackleg of ruminants caused by Clostridium chauvoei.

Development of a PCR technique specific for Demodex injai in biological specimens

The identification of Demodex injai as a second Demodex species of dog opened new questions and challenges in the understanding on the Demodex-host relationships. In this paper, we describe the development of a conventional PCR technique based on published genome sequences of D. injai from GenBank that specifically detects DNA from D. injai. This technique amplifies a 238-bp fragment corresponding to a region of the mitochondrial 16S rDNA of D. injai. The PCR was positive in DNA samples obtained from mites identified morphologically as D. injai, which served as positive controls, as well as in samples from three cases of demodicosis associated with proliferation of mites identified as D. injai. Furthermore, the PCR was positive in 2 out of 19 healthy dogs. Samples of Demodex canis and Demodex folliculorum were consistently negative. Skin samples from seven dogs with generalized demodicosis caused by D. canis were all negative in the D. injai-specific PCR, demonstrating that in generalized canine demodicosis, mite proliferation is species-specific. This technique can be a useful tool in the diagnosis and in epidemiologic and pathogenic studies.

Species-specific diagnostic assays for Bonamia ostreae and B. exitiosa in European flat oyster Ostrea edulis: conventional, real-time and multiplex PCR

Bonamia ostreae and B. exitiosa have caused mass mortalities of various oyster species around the world and co-occur in some European areas. The World Organisation for Animal Health (OIE) has included infections with both species in the list of notifiable diseases. However, official methods for species-specific diagnosis of either parasite have certain limitations. In this study, new species-specific conventional PCR (cPCR) and real-time PCR techniques were developed to diagnose each parasite species. Moreover, a multiplex PCR method was designed to detect both parasites in a single assay. The analytical sensitivity and specificity of each new method were evaluated. These new procedures were compared with 2 OIE-recommended methods, viz. standard histology and PCR-RFLP. The new procedures showed higher sensitivity than the OIE recommended ones for the diagnosis of both species. The sensitivity of tests with the new primers was higher using oyster gills and gonad tissue, rather than gills alone. The lack of a 'gold standard' prevented accurate estimation of sensitivity and specificity of the new methods. The implementation of statistical tools (maximum likelihood method) for the comparison of the diagnostic tests showed the possibility of false positives with the new procedures, although the absence of a gold standard precluded certainty. Nevertheless, all procedures showed negative results when used for the analysis of oysters from a Bonamia-free area.

Rabex-5 Protein Regulates Dendritic Localization of Small GTPase Rab17 and Neurite Morphogenesis in Hippocampal Neurons

Small GTPase Rab17 has recently been shown to regulate dendritic morphogenesis of mouse hippocampal neurons; however, the exact molecular mechanism of Rab17-mediated dendritogenesis remained to be determined, because no guanine nucleotide exchange factor (GEF) for Rab17 had been identified. In this study we screened for the Rab17-GEF by performing yeast two-hybrid assays with a GDP-locked Rab17 mutant as bait and found that Rabex-5 and ALS2, both of which were originally described as Rab5-GEFs, interact with Rab17. We also found that expression of Rabex-5, but not of ALS2, promotes translocation of Rab17 from the cell body to the dendrites of developing mouse hippocampal neurons. The shRNA-mediated knockdown of Rabex-5 or its known downstream target Rab5 in hippocampal neurons inhibited morphogenesis of both axons and dendrites, whereas knockdown of Rab17 affected dendrite morphogenesis alone. Based on these findings, we propose that Rabex-5 regulates neurite morphogenesis of hippocampal neurons by activating at least two downstream targets, Rab5, which is localized in both axons and dendrites, and Rab17, which is localized in dendrites alone.

Real-Time Polymerase Chain Reaction Versus Conventional PCR: A Comparison between two Methods for the Detection of Fusobacterium Nucleatum in Saliva, Nasopharyngeal Secretion and Middle Ear Effusion Samples

ABSTRACTOral and middle ear infections are biofilm related infections. Fusobacterium nucleatum is considered to be a key species in dental biofilm formation. The aim of this study was to compare conventional and real-time PCR, for the detection of F. nucleatum in saliva, nasopharyngeal secretion and middle ear effusion samples. Sixty samples were collected from 20 patients with otitis media with effusion. Species-specific 16S rRNA primers were used for conventional PCR and species-specific primers and TaqMan probes were used for real-time PCR analysis. Real-time PCR had a better performance than conventional PCR in detecting bacteria in the analyzed samples. The tested real-time PCR assay can identify and quantify F. nucleatum in biofilm samples with a high degree of sensitivity when compared with conventional PCR techniques.

Noninvasive prenatal fetal genotyping from cell-free maternal plasma

Fetal cell-free DNA circulating in maternal plasma is a possible target for various prenatal investigations of the unborn child. Typing of autosomal and gonosomal short tandem repeat and insertion-deletion polymorphisms was carried out in two pregnancies. Competitive PCR was tested versus allele and haplotype specific PCR by conventional and real-time PCR techniques.

Clinical and Microbiological Periodontal Evaluation of Mothers of Preterm Babies: A Case-Control Study

Objective: To establish the periodontal clinical parameters and frequency of periodontopathogenic bacteria in the subgingival biofilms of mothers of preterm babies compared with the mothers of term babies. Method: A case-control study involving 40 women was performed. In the study group were included 20 mothers of preterm babies, while the control group had 20 mothers of term babies. Within 48 h post birth, the subjects were interviewed for collecting information about identification, sociodemographic data, day-to-day habits, current and former gestational history, and periodontal examination (periodontal probing depth, bleeding on probing, plaque index and loss of periodontal attachment, and subgingival biofilm collection). The microbiological analysis of the biofilm detected the bacteria Tannerella forsythia, Treponema denticola, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans by the conventional PCR technique. Data were analyzed by the Student’s t-test with unequal variances, Pearson’s chi square test or Fisher’s exact test when the conditions for use of the chi square test were not fulfilled. The significance level was set at 5% and the confidence interval at 95%. Results: The means for clinical attachment loss and bleeding on probing were significantly greater among the mothers of preterm babies compared with mothers of term babies (p=0.049 and p=0.031, respectively). Porphyromonas gingivalis was significantly more frequent in the case group (p=0.044). Conclusion: It may be suggested that periodontal inflammation and attachment loss, as well as the presence of Porphyromonas gingivalis in the subgingival biofilms of the mothers of preterm babies can be associated with the preterm birth of the children.

Molecular Characterization of Transgene Integration by Next-Generation Sequencing in Transgenic Cattle

As the number of transgenic livestock increases, reliable detection and molecular characterization of transgene integration sites and copy number are crucial not only for interpreting the relationship between the integration site and the specific phenotype but also for commercial and economic demands. However, the ability of conventional PCR techniques to detect incomplete and multiple integration events is limited, making it technically challenging to characterize transgenes. Next-generation sequencing has enabled cost-effective, routine and widespread high-throughput genomic analysis. Here, we demonstrate the use of next-generation sequencing to extensively characterize cattle harboring a 150-kb human lactoferrin transgene that was initially analyzed by chromosome walking without success. Using this approach, the sites upstream and downstream of the target gene integration site in the host genome were identified at the single nucleotide level. The sequencing result was verified by event-specific PCR for the integration sites and FISH for the chromosomal location. Sequencing depth analysis revealed that multiple copies of the incomplete target gene and the vector backbone were present in the host genome. Upon integration, complex recombination was also observed between the target gene and the vector backbone. These findings indicate that next-generation sequencing is a reliable and accurate approach for the molecular characterization of the transgene sequence, integration sites and copy number in transgenic species.

Application and evaluation of a molecular approach for detection of the schistosomicidal effect of Mirazid® (myrrh) in the murine model

The conventional PCR technique was used for studying the schistosomicidal effect of Mirazid® in the murine model. Results of the molecular study were compared with the parasitological results (ova and worm count). The used PCR technique was more sensitive than the Kato-Katz thick smears. Mirazid® showed some schistosomicidal effects against murine Schistosoma mansoni. However, it was not efficient enough to cure any of the studied mice.