Duplex PCR Assay Research Articles

Development of Duplex PCR Assay for Detection and Differentiation of Typical and Atypical <i>Melissococcus plutonius</i> strains

ABSTRACTMelissococcus plutonius is the causative agent of an important honeybee disease, European foulbrood (EFB). In addition to M. plutonius strains with typical characteristics (typical M. plutonius), we recently reported the presence of atypical M. plutonius, which are phenotypically and genetically distinguished from typical M. plutonius. Because typical and atypical M. plutonius may have different pathogenic mechanisms, differentiation of these two types is very important for diagnosis and more effective control of EFB. In this study, therefore, a duplex PCR assay was developed to detect and differentiate typical and atypical M. plutonius rapidly and easily. On the basis of the results of comparative genomic analyses, we selected Na+/H+ antiporter gene and Fur family transcriptional regulator gene as targets for detection of typical and atypical strains, respectively, by PCR. Under optimized conditions, the duplex PCR system using the designed primers successfully detected and differentiated all typical and atypical M. plutonius strain/isolates tested, while no product was generated from any other bacterial strains/isolates used in this study, including those isolated from healthy honeybee larval guts. Detection limits of the PCR were 50 copies of chromosome/reaction for both types, and it could detect typical and atypical M. plutonius directly from diseased honeybee larvae. Moreover, the duplex PCR diagnosed mixed infections with both M. plutonius types more precisely than standard culture methods. These results indicate that the duplex PCR assay developed in this study is extremely useful for precise diagnosis and epidemiological study of EFB.

Rapid Differentiation between Livestock-Associated and Livestock-Independent Staphylococcus aureus CC398 Clades

Staphylococcus aureus clonal complex 398 (CC398) isolates cluster into two distinct phylogenetic clades based on single-nucleotide polymorphisms (SNPs) revealing a basal human clade and a more derived livestock clade. The scn and tet(M) genes are strongly associated with the human and the livestock clade, respectively, due to loss and acquisition of mobile genetic elements. We present canonical single-nucleotide polymorphism (canSNP) assays that differentiate the two major host-associated S. aureus CC398 clades and a duplex PCR assay for detection of scn and tet(M). The canSNP assays correctly placed 88 S. aureus CC398 isolates from a reference collection into the human and livestock clades and the duplex PCR assay correctly identified scn and tet(M). The assays were successfully applied to a geographically diverse collection of 272 human S. aureus CC398 isolates. The simple assays described here generate signals comparable to a whole-genome phylogeny for major clade assignment and are easily integrated into S. aureus CC398 surveillance programs and epidemiological studies.

Rapid Differentiation of Cryptic Species within Cryptococcus gattii by a Duplex PCR Assay

Cryptococcus gattii consists of four cryptic species, VGI, VGII, VGIII, and VGIV. Herein, a duplex PCR assay using two primer pairs targeting the vacuolar membrane gene and the intergenic spacer region was developed. It successfully distinguished the cryptic species according to the distinct size of the amplicons.

Uniplex and duplex PCR detection of geminivirus associated with potato apical leaf curl disease in India

Apical leaf curl disease has emerged as a new disease in potato during the last decade in India due to a change in planting date and an increased whitefly population. Its incidence is on the rise threatening the cultivation of potato across the country. Hence, a PCR assay was developed for the detection of Tomato leaf curl New Delhi virus-potato (ToLCNDV-Potato) which is the causal agent of apical leaf curl disease in potato. Primers specific to the coat protein (AV1) and replicase (AC1) gene regions were designed and used for standardization of the PCR. Some of the primers (LCVCPF1/LCVCPR1, LCVREPF2/LCVREPR2, LCrep1F/LCrep2R) could detect the virus in 2.4–0.24pg of total DNA of infected plant. A duplex PCR assay was optimized with the selected coat protein gene specific primers and primers specific to potato urease gene, a housekeeping gene served as an internal check. The suitability of these primers was examined for detection of the virus in 80 potato apical leaf curl disease samples from 11 different potato growing states of India and also from micro-plants grown in tissue culture. The selected coat protein primer pair (LCVCPF1/LCVCPR1) was found to be conserved in all 80 isolates except for a few isolates, which had a single nucleotide substitution in the forward primer sequence. These substitutions did not interfere with amplification of the coat protein gene. The primers could detect the virus using a print-capture PCR assay both in the presence and absence of an internal control. These results indicate the robustness of the PCR assay for virus indexing of mother stocks in the seed production system.

Collected series of resected intraductal papillary mucinous tumours (IPMTs) of the pancreas and correlation between the mucin expression profile and the malignant potential

Collected series of resected intraductal papillary mucinous tumours (IPMTs) of the pancreas and correlation between the mucin expression profile and the malignant potential

The new variant of Chlamydia trachomatis was present as early as 2003 in Örebro County, Sweden, but remained undetected until 2006

ObjectivesIn 2006, a new variant of Chlamydia trachomatis (nvCT) was reported in Sweden. Because of a cryptic plasmid deletion, the nvCT was undetectable in several of the genetic diagnostic systems...

Taguchi optimization of duplex PCR for simultaneous identification ofStaphylococcus aureusandClostridium perfringensalpha toxins

Staphylococcus aureus and Clostridium perfringens are two major bacteria that infect open wounds and delay the healing process. The rapid and progressive deterioration of soft tissue during S.aureus and C.perfringens coinfections is due to analogous necrotic alpha toxins produced by the two organisms. The aim of this study was to determine the alpha toxins of S.aureus and C.perfringens by duplex PCR. The PCR assay employed two sets of primers: hlaf/r to amplify staphylococcal alpha toxin gene hla (274bp) and cpaf/r to amplify clostridial alpha toxin gene cpa (398bp) along with a competitive internal amplification control (608bp), simultaneously. Optimization of the duplex PCR assay was achieved by a modified Taguchi method, an engineering optimization process, in a nine-tube combinatorial array. The detection level of the duplex PCR was found to be 10pg of purified DNA or 10(3) CFUmL(-1) of S.aureus and 100pg of purified DNA or 10(4) CFUmL(-1) of C.perfringens. Other bacteria routinely found in tissue infections were tested for cross-reactivity and the duplex PCR turned out to be highly specific. This duplex PCR assay provides a rapid, robust and reliable alternative to the existing conventional techniques in establishing the aetiology of S.aureus and C.perfringens in soft tissue infections.

Detection of the A2058G and A2059G 23S rRNA Gene Point Mutations Associated with Azithromycin Resistance in Treponema pallidum by Use of a TaqMan Real-Time Multiplex PCR Assay

Macrolide treatment failure in syphilis patients is associated with a single point mutation (either A2058G or A2059G) in both copies of the 23S rRNA gene in Treponema pallidum strains. The conventional method for the detection of both point mutations uses nested PCR combined with restriction enzyme digestions, which is laborious and time-consuming. We initially developed a TaqMan-based real-time duplex PCR assay for detection of the A2058G mutation, and upon discovery of the A2059G mutation, we modified the assay into a triplex format to simultaneously detect both mutations. The point mutations detected by the real-time triplex PCR were confirmed by pyrosequencing. A total of 129 specimens PCR positive for T. pallidum that were obtained from an azithromycin resistance surveillance study conducted in the United States were analyzed. Sixty-six (51.2%) of the 129 samples with the A2058G mutation were identified by both real-time PCR assays. Of the remaining 63 samples that were identified as having a macrolide-susceptible genotype by the duplex PCR assay, 17 (27%) were found to contain the A2059G mutation by the triplex PCR. The proportions of macrolide-susceptible versus -resistant genotypes harboring either the A2058G or the A2059G mutation among the T. pallidum strains were 35.6, 51.2, and 13.2%, respectively. None of the T. pallidum strains examined had both point mutations. The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism analyses for the rapid detection of both point mutations associated with macrolide resistance in T. pallidum.

Validation of a commercial real-time PCR kit for specific and sensitive detection of Pseudorabies

Pseudorabies virus is the causative agent of Aujeszky's disease, one of the OIE listed diseases that mainly affects swine, but also can affect other animal species, and which can lead to heavy economic losses in pig industry. This study was designed to evaluate the performance of the ADIAVET(®) PRV REALTIME kit, a new commercial real time PCR kit for Pseudorabies virus genome detection developed by the French manufacturer Adiagène. It can be used on pig biological samples such as nasal swab supernatant, tonsil, brain or lung samples, or on samples from other susceptible animals, such as domestic carnivores. This ready-to-use duplex PCR assay contains an external positive control, appropriate for assessing DNA extraction efficiency and the presence of PCR inhibitors. The analytical specificity and sensitivity, intra- and inter-assay repeatability and diagnostic characteristics of the kit were determined and compared with virus isolation, which is the gold standard. Based on these results, the ADIAVET(®) PRV REALTIME kit received full validation for diagnostic purposes.

Performance comparison of phenotypic and molecular methods for detection and differentiation of Candida albicans and Candida dubliniensis

BackgroundCandida albicans is the most pathogenic Candida species but shares many phenotypic features with Candida dubliniensis and may, therefore, be misidentified in clinical microbiology laboratories. Candidemia cases due to C. dubliniensis are increasingly being reported in recent years. Accurate identification is warranted since mortality rates are highest for C. albicans infections, however, C. dubliniensis has the propensity to develop resistance against azoles more easily. We developed a duplex PCR assay for rapid detection and differentiation of C. albicans from C. dubliniensis for resource-poor settings equipped with basic PCR technology and compared its performance with three phenotypic methods.MethodsDuplex PCR was performed on 122 germ tube positive and 12 germ tube negative isolates of Candida species previously identified by assimilation profiles on Vitek 2 ID-YST system. Typical morphologic characteristics on simplified sunflower seed agar (SSA), and reaction with a commercial (Bichro-Dubli) latex agglutination test were also performed. The assay was further applied on 239 clinical yeast and yeast-like fungi and results were confirmed by DNA sequencing of internal transcribed spacer (ITS) region of rDNA.ResultsThe results of duplex PCR assay for 122 germ tube positive and 12 germ tube negative isolates of Candida species were comparable to their identification by Vitek 2 ID-YST system, colony characteristics on SSA and latex agglutination test. Application of duplex PCR also correctly identified all 148 C. albicans and 50 C. dubliniensis strains among 239 yeast-like fungi.ConclusionsThe data show that both, duplex PCR and Bichro-Dubli are reliable tests for rapid (within few hours) identification of clinical yeast isolates as C. dubliniensis or C. albicans. However, duplex PCR may be applied directly on clinical yeast isolates for their identification as C. dubliniensis or C. albicans as it does not require prior testing for germ tube formation or latex Candida agglutination.

‘Who's who’ in renal sphaerosporids (Bivalvulida: Myxozoa) from common carp, Prussian carp and goldfish – molecular identification of cryptic species, blood stages and new members of Sphaerospora sensu stricto

Myxozoans are a group of diverse, spore-forming metazoan microparasites bound to aquatic environments. Sphaerospora dykovae (previously S. renicola) causes renal sphaerosporosis and acute swim bladder inflammation (SBI) in juvenile Cyprinus carpio carpio, in central Europe. A morphologically similar species with comparably low pathogenicity, S. angulata has been described from C. c. carpio, Carassius auratus auratus and Carassius gibelio. To clarify uncertainties and ambiguities in taxon identification in these hosts we decided to re-investigate differences in spore morphology using a statistical approach, in combination with SSU and LSU rDNA sequence analyses. We found that developing spores of S. angulata and S. dykovae cannot be distinguished morphologically and designed a duplex PCR assay for the cryptic species that demonstrated S. dykovae is specific to C. c. carpio, whereas S. angulata infects C. a. auratus and C. gibelio. The molecular identification of myxozoan blood stages in common carp and goldfish, which had previously been ascribed to Sphaerospora spp. showed that approximately 75% of blood stages were from non-sphaerosporid coelozoic species infecting these cyprinids and more than 10% were from an alien species, Myxobilatus gasterostei, developing in sticklebacks. We hereby report non-selective myxozoan host invasion and multi-species infections, whose role in SBI still requires clarification.

Atypical cerebral listeriosis associated with Listeria innocua in a beef bull

Natural infections of cattle associated with Listeria innocua have not been reported. This report describes the first case of cerebral listeriosis in a bull due to Listeria innocua. The animal presented neurological signs characterized by weakness, incoordination and recumbency. Histopathologic evaluation of brain tissue revealed multifocal microabscesses, perivascular lymphocytic cuffing, vasculitis, oedema and haemorrhages. All lesions extended from the medulla oblongata to the basal nuclei/parietal cortex area. Indirect immunohistochemistry labelled for Listeria sp. in the brain tissue, but not for Listeria monocytogenes, neurotropic Flaviviruses, BVDV, bovine Herpesvirus 1, Chlamydophila spp. and Histophilus somni. PCR was negative for ovine herpesvirus. L. innocua was isolated from brainstem and identified by biochemical tests (Camp and beta-hemolysis negative). Subsequently, the species was confirmed by a duplex PCR and minisequencing assays. L. innocua should be histologically considered as a differential diagnosis of thrombotic meningoencephalitis, malignant catarrhal fever and cerebral listeriosis due to L. monocytogenes in cattle.

Evolutionary and experimental assessment of novel markers for detection of Xanthomonas euvesicatoria in plant samples.

BackgroundBacterial spot-causing xanthomonads (BSX) are quarantine phytopathogenic bacteria responsible for heavy losses in tomato and pepper production. Despite the research on improved plant spraying methods and resistant cultivars, the use of healthy plant material is still considered as the most effective bacterial spot control measure. Therefore, rapid and efficient detection methods are crucial for an early detection of these phytopathogens.MethodologyIn this work, we selected and validated novel DNA markers for reliable detection of the BSX Xanthomonas euvesicatoria (Xeu). Xeu-specific DNA regions were selected using two online applications, CUPID and Insignia. Furthermore, to facilitate the selection of putative DNA markers, a customized C program was designed to retrieve the regions outputted by both databases. The in silico validation was further extended in order to provide an insight on the origin of these Xeu-specific regions by assessing chromosomal location, GC content, codon usage and synteny analyses. Primer-pairs were designed for amplification of those regions and the PCR validation assays showed that most primers allowed for positive amplification with different Xeu strains. The obtained amplicons were labeled and used as probes in dot blot assays, which allowed testing the probes against a collection of 12 non-BSX Xanthomonas and 23 other phytopathogenic bacteria. These assays confirmed the specificity of the selected DNA markers. Finally, we designed and tested a duplex PCR assay and an inverted dot blot platform for culture-independent detection of Xeu in infected plants.SignificanceThis study details a selection strategy able to provide a large number of Xeu-specific DNA markers. As demonstrated, the selected markers can detect Xeu in infected plants both by PCR and by hybridization-based assays coupled with automatic data analysis. Furthermore, this work is a contribution to implement more efficient DNA-based methods of bacterial diagnostics.

Milk adulteration: Detection of bovine milk in bulk goat milk produced by smallholders in northeastern Brazil by a duplex PCR assay

The aim of this study was to investigate the adulteration of goat milk produced by smallholders in semiarid northeastern Brazil with bovine milk as an adulterant. The study was requested by the association of smallholder producers in the region to investigate and to inhibit adulteration practices as a need to ensure the quality and safety of goat milk. A duplex PCR assay has been developed and standardized. Further validation was performed in 160 fresh bulk goat milk samples. The detection limit of the duplex PCR was 0.5% bovine milk in goat milk and the results indicated that 41.2% of the goat milk presented to market was positive for bovine milk. Making the test available to the association of producers, together with extension activities, have been applied to reduce adulteration in goat milk sold to small-scale dairy plants and to ensure the species origin for goat milk in the state of Paraíba.

Molecular sexing of Neotropical otter (Lontra longicaudis) noninvasive samples

Noninvasive molecular assays allow the investigation of elusive, rare and/or endangered organisms, and are useful for estimates of population parameters. Sex determination of noninvasive samples provides important information of sex ratio, social structure and dispersal patterns. Still, such assays remain unavailable for most carnivore species, precluding assessments of their population biology. Here we report a standardized protocol for molecular sexing of Neotropical otter noninvasive samples collected in the field. A duplex PCR assay was employed using a multiple-tube approach, resulting in reliable gender determination for 95% of the samples and presenting a low rate (<4%) of misassignment. This assay should foster the development of molecular ecological studies of this little-known species, and likely also contribute to similar endeavors on related otters.

A Duplex PCR Assay for Sex Determination of Cattle Meat by Simultaneous Amplification of SRY, AMELX and AMELY Genes

Analysis of the sex of cattle meat by fast and reliable molecular techniques is an important measure to ensure correct allocation of export refunds, particularly in European countries and also in countries such as India where female cattle (cow) slaughter is banned legally in many areas because of religious beliefs. Based on the male-specific SRY gene, X and Y chromosome specific amelogenin gene 2 pair of primers was designed and the system of PCR was optimized. Upon PCR amplification, male tissue showed 3 bands, while female tissue resulted in only 1 band. The accuracy and specificity of the primers were assessed using DNA template extracted from cattle meat of known sex. The protocol was subjected to a blind test and showed 100% concordance, proving its accuracy and reliability.

Erwinia amylovora Novel Plasmid pEI70: Complete Sequence, Biogeography, and Role in Aggressiveness in the Fire Blight Phytopathogen

Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5–92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora.

Comparison of DNA Extraction Methods and Development of Duplex PCR and Real-Time PCR To Detect Tomato, Carrot, and Celery in Food

Traceability is of particular importance for those persons who suffer allergy or intolerance to some food component(s) and need a strict avoidance of the allergenic food. In this paper, methodologies are described to fingerprint the presence of allergenic species such as carrot, tomato, and celery by DNA detection. Three DNA extraction methods were applied on vegetables and foods containing or not containing the allergens, and the results were compared and discussed. Fast SYBR Green DNA melting curve temperature analyses and duplex PCR assays with internal control have been developed for detection of these allergenic vegetables and have been tested on commercial foods. Spiking food experiments were also performed, assessing that limits of detection (LOD) of 1 mg/kg for carrot and tomato DNA and 10 mg/kg for celery DNA have been reached.

Duplex PCR assay for the detection of avian adeno virus and chicken anemia virus prevalent in Pakistan

Avian Adeno viruses and Chicken Anemia Viruses cause serious economic losses to the poultry industry of Pakistan each year. Timely and efficient diagnosis of the viruses is needed in order to practice prevention and control strategies. In the first part of this study, we investigated broilers, breeder and Layer stocks for morbidity and mortality rates due to AAV and CAV infections and any co-infections by examining signs and symptoms typical of their infestation or post mortem examination. In the second part of the study, we developed a duplex PCR assay for the detection of AAV and CAV which is capable to simultaneously detect both the viral types prevalent in Pakistan with high sensitivity and 100% specificity.

A Suitable Duplex PCR for Ovine Embryo Sex and Genotype of PrnP Gene Determination for MOET-Based Selection Programmes

The objective of this study was to test the suitability of a duplex PCR assay for sex and scrapie resistance genotype determination in fresh embryos. Duplex PCR amplified a repetitive and specific fragment of Y chromosome, used for sex diagnosis, and a PrnP fragment. PrnP codons 134 and 156, and codon 171 were genotyped by restriction fragment length polymorphisms and allele-specific PCR, respectively, after re-amplification of PrnP fragment. The specificity of the method was first assessed by testing 359 blood samples from Rasa Aragonesa sheep breed (161 males and 198 females). No amplification failures and total agreement between genotypic and phenotypic sex were found. In the same way, PrnP genotype determination by duplex PCR assay was in agreement with the PrnP animal's genotype established by sequencing. Finally, 73 samples of 1-10 cells from compact morulae were aspirated through the zona pellucida and genotyped for sex and PrnP. The efficiency was 96% when three or more cells were sampled. These results confirm that the duplex PCR assay reported in this work can be used for rapid sex determination in ovine embryos, with a high efficiency and accuracy (96%) when three or more cells are sampled, allowing sexed fresh embryos of known PrnP genotype to be transferred in multiple ovulation and embryo transfer programmes.