Sensitive Multiplex Real-time PCR Research Articles

Suitability of NIID-MDCK cells as a substrate for cell-based influenza vaccine development from the perspective of adventitious virus susceptibility.

The practical use of cell-based seasonal influenza vaccines is currently being considered in Japan. From the perspective of adventitious virus contamination, we assessed the suitability of NIID-MDCK cells (NIID-MDCK-Cs) as a safe substrate for the isolation of influenza viruses from clinical specimens. We first established a sensitive multiplex real-time PCR system to screen for 27 respiratory viruses and used it on 34 virus samples that were isolated by passaging influenza-positive clinical specimens in NIID-MDCK-Cs. Incidentally, the limit of detection (LOD) of the system was 100 or fewer genome copies per reaction. In addition to influenza viruses, human enterovirus 68 (HEV-D68) genomes were detected in two samples after two or three passages in NIID-MDCK-Cs. To further investigate the susceptibility of NIID-MDCK-Cs to adventitious viruses, eight common respiratory viruses were subjected to passages in NIID-MDCK-Cs. The genome copy numbers of seven viruses other than parainfluenza 3 decreased below the LOD by passage 4. By passaging in NIID-MDCK-Cs, the genome numbers of the input HEV-D68, 1 × 108 copies, declined to 102 at passage 3 and to under the LOD at passage 4, whereas those of the other six viruses were under the LOD by passage 3. These results implied that during the process of isolating influenza viruses with NIID-MDCK-Cs, contaminating viruses other than parainfluenza 3 can be efficiently removed by passages in NIID-MDCK-Cs. NIID-MDCK-Cs could be a safe substrate for isolating influenza viruses that can be used to develop cell-based influenza vaccine candidate viruses.

Monitoring of HTLV-1-associated diseases by proviral load quantification using multiplex real-time PCR.

Proviral load (PVL) is one of the determining factors for the pathogenesis and clinical progression of the human T-lymphotropic virus type I (HTLV-1) infection. In the present study, we optimized a sensitive multiplex real-time PCR for the simultaneous detection and quantification of HTLV-1 proviral load and beta-globin gene as endogenous control. The values obtained for HTLV-1 PVL were used to monitor the clinical evolution in HTLV-1-infected individuals. A vector containing cloned DNA targets of the real-time PCR for the beta-globin gene and the HTLV-1pol region was constructed. For the reaction validation, we compared the amplification efficiency of the constructed vector and MT-2 cell line containing HTLV-1. The analytical sensitivity of the reaction was evaluated by the application of a standard curve with a high order of magnitude. PVL assay was evaluated on DNA samples of HTLV-1 seropositive individuals. The construct showed adequate amplification for the beta-globin and HTLV-1 pol genes when evaluated as multiplex real-time PCR (slope = 3.23/3.26, Y-intercept = 40.18/40.73, correlation coefficient r2 = 0.99/0.99, and efficiency = 103.98/102.78, respectively). The quantification of PVL using the MT-2 cell line was equivalent to the data obtained using the plasmidial curve (2.5 copies per cell). In HTLV-1-associatedmyelopathy/tropical spastic paraparesis patients, PVL was significantly higher (21315 ± 2154 copies/105 PBMC) compared to asymptomatic individuals (1253 ± 691 copies/105 PBMC). The obtained results indicate that the optimized HTLV-1 PVL assay using plasmidial curve can be applied for monitoring and follow-up of the progression of HTLV-1 disease. The use of a unique reference plasmid for both HTLV-1 and endogenous gene allows a robust and effective quantification of HTLV-1 PVL. In addition, the developed multiplex real-time PCR assay was efficient to be used as a tool to monitor HTLV-1-infected individuals.

Molecular Detection of Ehrlichia canis in the Pet-Dog Population in R. N. Macedonia

Abstract Canine monocytic ehrlichiosis (CME) is a widespread, tick-borne, canine disease, caused by an obligate intracellular bacterium, Ehrlichia canis. The main vector, a brown-dog tick, Rhipicephalus sanguineus, is widely distributed, especially in areas with tropic, subtropic, or Mediterranean climates (Central and South America, Eastern and Western Asia, Africa, Australia and Southern Europe). The study performed in 2012, by Stefanovska et al., determined a seroprevalence of 18.7% of E. canis among the Macedonian dog population. Up to date, the presence of E. canis, using molecular diagnostic methods, has not been investigated in Macedonia. Therefore, this study aimed to confirm the presence of E. canis, in the pet-dog population on the territory of the city of Skopje, North Macedonia, using a highly sensitive multiplex Real-Time PCR method (qPCR). Whole blood samples from 80 dogs of different breeds and ages, with clinical symptoms of CME and positive serology result for the presence of antibodies against E.canis, were collected for analyses. Out of 80 dogs, 36 (45%) were found as positive. The present work reports the first molecular detection of E. canis in pet dogs on the territory of the city of Skopje, Macedonia.

A fast and sensitive multiplex real-time PCR assay for simultaneous identification of Bursaphelenchus xylophilus, B. mucronatus and B. fraudulentus – three closely related species from the xylophilus group

The quarantine nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease in Asia and Europe. Differentiation between B. xylophilus and other related, non-pathogenic species can be ambiguous when based exclusively on morphological characters. The morphology of B. mucronatus and B. fraudulentus most closely resembles that of B. xylophilus. Moreover, all of these nematodes are found in both Asia and Europe and can colonise various species of pine. Therefore, for phytosanitary purposes it is necessary to identify the three species precisely and rapidly. In this paper we described the results of the optimized multiplex real-time PCR protocol that utilizes two universal primers and three specific TaqMan probes to identify and differentiate the three Bursaphelenchus species, simultaneously. Species-specific real-time PCR reactions gave expected products for B. xylophilus, B. mucronatus and B. fraudulentus templates. The optimized primer combination together with designed species-specific probes has produced reliable results in multiplex PCR assays with eleven geographically distant isolates of B. xylophilus, eleven of B. mucronatus, seven of B. fraudulentus, and no cross-reactions were observed between tested samples and with other species (i.e., B. piniperdae, B. pinophilus, B. populi and Parasitaphelenchus papillatus). All species-specific real-time PCR reactions performed on DNA extracted from a single nematode individual consistently gave specific amplicons, confirming the reproducibility of the method. Moreover, the conducted study revealed that 30 fg was the lowest DNA input still detected in multiplex real-time PCR analysis. The described approach is simple, time-saving, and reliable in comparison to conventional PCR, and it can be used for simultaneous identification of the above three species within the xylophilus group.

Fast Multiplex real time PCR method for sex-identification of medaka (Oryzias latipes) by non-invasive sampling

Medaka fish (Oryzias latipes) has been widely used in fish screening and multi-generation tests to provide relevant data to assess impacts of endocrine disrupting chemicals (EDCs) in fish populations.The genotypic differentiation of Medaka sex allows diagnosing the sex reversal, and is required in current test guidelines (e.g. OECD TG 240, 2015). DNA isolation for genetic sex-identification requires sample collection, which has been normally conducted using invasive (fish sacrifice) or semi-invasive (fin-clip) procedures, which conflicts with the need for a fast, simple, and stress-free method. Swabbing skin mucus to collect DNA has been adopted in ecological studies of larger fish, however for smaller fish, it has to be established.To handle larger number of samples, real-time PCR represents a faster and sensitive method compared to conventional PCR. In this study, we aimed to develop a multiplex real-time PCR method for Medaka genetic sex-identification, using DNA sampled by swabbing as less invasive technique. In this approach, the male-determining gene DMY was used in combination with the cytochrome b housekeeping gene.•The method developed is a robust, rapid and a sensitive multiplex real-time PCR for Medaka genetic sex-identification.•This method allows the use of DNA isolated from fish by swabbing, as non-invasive sampling method.

Simultaneous identification of Haemophilus influenzae and Haemophilus haemolyticus using real-time PCR.

To design a highly specific and sensitive multiplex real-time PCR assay for the differentiation of the pathogen Haemophilus influenzae from its nonpathogenic near-neighbor Haemophilus haemolyticus. A comparison of 380 Haemophilus spp. genomes was used to identify loci specific for each species. Novel PCR assays targeting H. haemolyticus (hypD) and H. influenzae (siaT) were designed. PCR screening across 143 isolates demonstrated 100% specificity for hypD and siaT. These two assays were multiplexed with the recently described fucP assay for further differentiation among H. influenzae. The triplex assay provides rapid, unambiguous, sensitive and highly specific genotyping results for the simultaneous detection of hypD and siaT, including fucose-positive H. influenzae (fucP), in a single PCR.

Sensitive Molecular Diagnostics for Cutaneous Leishmaniasis

BackgroundRapid diagnosis of cutaneous leishmaniasis (CL) and identification of Leishmania species is highly important for the disease management. In Israel, CL is caused mainly by Leishmania major and Leishmania tropica species.MethodsWe established an easy to handle point of care lesion-swabbing, combined with a highly sensitive multiplex real time PCR (multiplex qPCR) for accurate and rapid diagnosis of Leishmania species.ResultsUsing three probes: one general for: Leishmania species, and two specific for L major, and L tropica, we screened 1783 clinical samples collected during two years. Leishmania species was found in 1086 individuals, 1008 L major, and 70 L tropica. Eight samples positive for Leishmania species only, were further tested using a second set of multiplex qPCR developed, and were found positive for Leishmania braziliensis and Leishmania infantum/donovani (2 and 6 samples, concomitantly).ConclusionsTaken together, the test enabled diagnostics and better treatment of Leishmania infections from the Old World (1078 samples) and the New World (8 samples), and the subtyping of the dominant strains in the region, as well as in returning travelers’.

A sensitive multiplex real-time PCR panel for rapid diagnosis of viruses associated with porcine respiratory and reproductive disorders

The objective of this study was to develop a multiplex real-time PCR panel using TaqMan probes for the detection and differentiation of porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus North American type (PRRSV-NA), pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine parvovirus type 1 (PPV1) and Japanese encephalitis virus (JEV). Specific primer and probe combinations for PCV2, PRRSV, PRV, CSFV, PPV1 and JEV were selected within the conserved region of each viral genome. The multiplex real-time PCR panel which was run in two separate tubes was capable of specific detection of the six selected pig viruses, without cross-reactions with other non-targeted pig viruses. The detection limit of the assays was 10 copies/μL for PCV2, PRV, CSFV and PRRSV and 100 copies/μL for PPV and JEV. The two-tube multiplex real-time PCR panel showed 99.2% concordance with conventional PCR assays on 118 field samples. Overall, the multiplex real-time PCR panel provides a fast, specific, and sensitive diagnostic tool for detection of multiple viral pathogens in pigs and will be useful not only for diagnostics, or ecological, epidemiological and pathogenesis studies, but also for investigating host/virus or virus/virus interactions, particularly during coinfections.

Development of multiplex real-time PCR with Internal amplification control for simultaneous detection of Salmonella and Cronobacter in powdered infant formula

Contamination of powdered infant formula (PIF) by the bacteria Cronobacter spp. and Salmonella enterica was deemed a matter of great concern by the World Health Organization and the Food and Agriculture Organization of the United Nations in 2004. Therefore, we developed a rapid and sensitive multiplex real-time PCR assay for the simultaneous detection of Cronobacter and Salmonella in PIF. In addition, an internal amplification control (IAC) was also included for exclusion of false negative results in this study. The quantitative detection range for pure cultures in this optimized multiplex real-time PCR assay was 10 3 to 10 8 CFU/ml for both Salmonella and Cronobacter. When our established multiplex real-time PCR system was applied to artificially contaminated PIF, the detection limit was 10 3 CFU/ml for Salmonella and Cronobacter without enrichment. The commercial PIF was then inoculated with Salmonella and Cronobacter at 10, 1 and 0.1 CFU per gram of formula and the single enrichment broth samples were analyzed by multiplex real-time PCR after enrichment for 9, 12, and 24 h. At 12 h post-enrichment, we could detect Salmonella and Cronobacter at initial inoculation levels of approximately 0.1 CFU/g in PIF. Additionally, stable fluorescent IAC signals could be assessed between 29 and 34 cycles of PCR amplification. Results from this study showed that the multiplex real-time PCR assay is an effective method for the rapid and simultaneous detection and quantification of Cronobacter and Salmonella in PIF .

Detection of a broad range of human adenoviruses in respiratory tract samples using a sensitive multiplex real‐time PCR assay

Human adenoviruses (hAdVs) are associated with acute respiratory tract infections in pediatric populations and have been identified as a cause of outbreaks in institutional settings. Rapid diagnosis of hAdV infection is critical for appropriate and timely management. This study reports the design and validation of a sensitive and specific multiplex real‐time PCR for the detection of a broad range of hAdV serotypes in respiratory samples. The assay targets the conserved region of the hAdV hexon gene and utilizes hydrolysis probes for the detection of amplified products. The assay was evaluated using retrospectively (n = 864) and prospectively (n = 11,451) collected samples from November 2005 to July 2006. Seasonality studies and analysis of outbreaks was conducted over a 2‐year period from January 2005 to December 2006 (n = 33,067 samples). The assay gave a hAdV positive rate of 7.1% (n = 811) for specimens tested prospectively and was able to detect a broad range of hAdV serotypes with good sensitivity and specificity. A high rate of co‐infection was noted (21.7%). Adenovirus infections were more prevalent in the young with a median age of 24 months for positive patients. Sequence analysis of hAdV positives showed that serotype 7 was the most prevalent followed by serotypes 2 and 3. Association of hAdVs with respiratory outbreaks was low at 2.3% (6 of 266 outbreaks tested) and no seasonal variation was observed for hAdV infections during the 2‐year study period. This assay can improve the detection of hAdVs in respiratory samples and can be used to provide valuable epidemiological information. J. Med. Virol. 80:856–865, 2008. © 2008 Wiley‐Liss, Inc.