Real-time PCR Assay For Detection Research Articles

Optimization and Evaluation of Triplex Real-time PCR Assay for Detection of Genes Encoding Staphylococcal Virulence and Methicillin Resistance Using Two Different Multi-channel Emission Instruments

Optimization and Evaluation of Triplex Real-time PCR Assay for Detection of Genes Encoding Staphylococcal Virulence and Methicillin Resistance Using Two Different Multi-channel Emission Instruments

Development of Real-time PCR Assay for Detection of Rotavirus Infection in Diarrheic Bovine Calves

Development of Real-time PCR Assay for Detection of Rotavirus Infection in Diarrheic Bovine Calves

Specific PCR and real-time PCR assays for detection and quantitation of ‘Candidatus Phytoplasma phoenicium’

Almond witches' broom (AlmWB) is a fast-spreading lethal disease of almond, peach and nectarine associated with ‘Candidatus Phytoplasma phoenicium’. The development of PCR and quantitative real-time PCR (qPCR) assays for the sensitive and specific detection of the phytoplasma is of prime importance for early detection of ‘Ca. P. phoenicium’ and for epidemiological studies. The developed qPCR assay herein uses a TaqMan® probe labeled with Black Hole Quencher Plus. The specificity of the PCR and that of the qPCR detection protocols were tested on 17 phytoplasma isolates belonging to 11 phytoplasma 16S rRNA groups, on samples of almond, peach, nectarine, native plants and insects infected or uninfected with the phytoplasma. The developed assays showed high specificity against ‘Ca. P. phoenicium’ and no cross-reactivity against any other phytoplasma, plant or insect tested. The sensitivity of the developed PCR and qPCR assays was similar to the conventional nested PCR protocol using universal primers. The qPCR assay was further validated by quantitating AlmWB phytoplasma in different hosts, plant parts and potential insect vectors. The highest titers of ‘Ca. P. phoenicium’ were detected in the phloem tissues of stems and roots of almond and nectarine trees, where they averaged from 105 to 106 genomic units per nanogram of host DNA (GU/ng of DNA). The newly developed PCR and qPCR protocols are reliable, specific and sensitive methods that are easily applicable to high-throughput diagnosis of AlmWB in plants and insects and can be used for surveys of potential vectors and alternative hosts.

Multi-azole-resistant Aspergillus fumigatus in the environment in Tanzania.

Azole resistance in Aspergillus fumigatus isolates has been increasingly reported with variable prevalence worldwide and is challenging the effective management of aspergillosis. Here we report the coexistence of both TR₃₄/L98H and TR₄₆/Y121F/T289A resistance mechanisms in azole-resistant A. fumigatus (ARAF) isolates originating from Tanzania, Africa. A total of 30 soil and woody debris samples from the surroundings of Kilimanjaro Christian Medical Centre, Moshi, Tanzania, were processed for detection of ARAF isolates and were investigated for susceptibility to itraconazole, voriconazole, posaconazole and isavuconazole. All ARAF isolates were subjected to a real-time PCR assay for detection of mutations and were genotyped by microsatellite typing. Of the 30 samples, 29 yielded 108 A. fumigatus isolates. Overall, 15 ARAF isolates were obtained, which included 4 ARAF harbouring the TR₄₆/Y121F/T289A mutation and 11 isolates carrying TR₃₄/L98H. All four TR₄₆/Y121F/T289A A. fumigatus isolates showed high MICs of voriconazole (>16 mg/L) and isavuconazole (8 mg/L). In contrast, the 11 TR₃₄/L98H A. fumigatus isolates were pan-azole resistant. The isolates were cross-resistant to azole fungicides. Notably, 20% of environmental samples harboured ARAF and the TR₄₆/Y121F/T289A resistance mechanism was found in 5.5% of the soil samples, where it coexisted with TR₃₄/L98H. The Tanzanian TR₄₆/Y121F/T289A strains had a genotype identical to Dutch clinical TR₄₆/Y121F/T289A isolates. The present study reports the isolation of resistant A. fumigatus strains harbouring the TR₄₆/Y121F/T289A mutation from Africa. Recovery of TR₄₆/Y121F/T289A from the environment is worrisome and we must strive for effective surveillance of clinical and environmental sources to detect azole resistance in A. fumigatus.

Clinical validation of a multiplex real-time PCR assay for detection of invasive candidiasis in intensive care unit patients.

New techniques, such as those based on multiplex quantitative real-time PCR (MRT-PCR), can improve the detection of invasive candidiasis (IC). We prospectively studied 63 intensive care unit patients with suspected IC and 40 healthy controls. Blood cultures and MRT-PCR were performed at day 0 and +2, +7, +14 and +21 days in all patients. In addition, β-d-glucan (BDG) and Candida albicans germ tube antibody (CAGTA) were quantified. IC was confirmed in 27 patients. Colonization was significantly higher in patients with IC (96% versus 64%, P = 0.002). The sensitivity, specificity, positive predictive value and negative predictive value of MRT-PCR for the diagnosis of IC were 96.3%, 97.3%, 92.8% and 98.7%, respectively. The positive predictive value and specificity were significantly higher for MRT-PCR than for BDG and CATGA. MRT-PCR performed very well, especially in deep-seated IC (sensitivity 90.9% versus 45.4% for blood culture; P = 0.06). As regards the most appropriate clinical sample for DNA amplification, in this study whole blood and serum presented similar results. MRT-PCR appears to be a useful test for confirming a diagnosis of IC in critically ill patients, especially in those with deep-seated disease. Its high sensitivity and positive predictive value make it a much more efficient tool for the management of IC than other diagnostic procedures and clinical scores.

Performance of 3 real-time PCR assays for direct detection of Staphylococcus aureus and MRSA from clinical samples

We compared 3 real-time PCR assays: off-label use of 2 commercial assays (BD-GeneOhm™ MRSA assay for methicillin-resistant Staphylococcus aureus [MRSA] detection and BD-GeneOhm StaphSR™ for MRSA and methicillin-susceptible S. aureus detection) and an in-house real-time PCR assay for detection of total S. aureus from clinical specimens. Testing was performed on 200 distinct specimens. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated using culture as the gold standard. The prevalence of S. aureus in the samples was 44.5%, and MRSA was 20%. For total S. aureus, the StaphSR-PCR and the in-house PCR assays had a sensitivity and specificity of 94.4% and 96.4% and 93.3% and 99.1%, respectively. For MRSA detection, the StaphSR and the BD GeneOhm assay had a sensitivity and specificity of 92.5% and 98.8% and 92.5% and 96.3%, respectively. This study demonstrates the potential use of tests like the StaphSR-PCR assay for rapid detection of S. aureus and MRSA directly from clinical specimens; however, culture follow-up would be needed to identify other potential pathogens in the specimen.

Multiplex real-time PCR assays for detection of four seedborne spinach pathogens

To develop multiplex TaqMan real-time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach. Primers and probes were designed from conserved sequences of the internal transcribed spacer (for Peronospora farinosa f. sp. spinaciae and Stemphylium botryosum), the intergenic spacer (for Verticillium dahliae) and the elongation factor 1 alpha (for Cladosporium variabile) regions of DNA. The TaqMan assays were tested on DNA extracted from numerous isolates of the four target pathogens, as well as a wide range of nontarget, related fungi or oomycetes and numerous saprophytes commonly found on spinach seed. Multiplex real-time PCR assays were evaluated by detecting two or three target pathogens simultaneously. Singular and multiplex real-time PCR assays were also applied to DNA extracted from bulked seed and single spinach seed. The real-time PCR assays were species-specific and sensitive. Singular or multiplex real-time PCR assays could detect target pathogens from both bulked seed samples as well as single spinach seed. The freeze-blotter assay that is currently routinely used in the spinach seed industry to detect and quantify three fungal seedborne pathogens of spinach (C.variabile, S.botryosum and V.dahliae) is quite laborious and takes several weeks to process. The real-time PCR assays developed in this study are more sensitive and can be completed in a single day. As the assays can be applied easily for routine seed inspections, these tools could be very useful to the spinach seed industry.

Development and Evaluation of a Real-Time PCR Assay for Detection of Pneumocystis jirovecii on the Fully Automated BD MAX Platform

Development and Evaluation of a Real-Time PCR Assay for Detection of Pneumocystis jirovecii on the Fully Automated BD MAX Platform

Real-time PCR assays for detection of Brucella spp. and the identification of genotype ST27 in bottlenose dolphins (Tursiops truncatus)

Rapid detection of Brucella spp. in marine mammals is challenging. Microbiologic culture is used for definitive diagnosis of brucellosis, but is time consuming, has low sensitivity and can be hazardous to laboratory personnel. Serological methods can aid in diagnosis, but may not differentiate prior exposure versus current active infection and may cross-react with unrelated Gram-negative bacteria. This study reports a real-time PCR assay for the detection of Brucella spp. and application to screen clinical samples from bottlenose dolphins stranded along the coast of South Carolina, USA. The assay was found to be 100% sensitive for the Brucella strains tested, and the limit of detection was 0.27fg of genomic DNA from Brucella ceti B1/94 per PCR volume. No amplification was detected for the non-Brucella pathogens tested. Brucella DNA was detected in 31% (55/178) of clinical samples tested. These studies indicate that the real-time PCR assay is highly sensitive and specific for the detection of Brucella spp. in bottlenose dolphins. We also developed a second real-time PCR assay for rapid identification of Brucella ST27, a genotype that is associated with human zoonotic infection. Positive results were obtained for Brucella strains which had been identified as ST27 by multilocus sequence typing. No amplification was found for other Brucella strains included in this study. ST27 was identified in 33% (18/54) of Brucella spp. DNA-positive clinical samples. To our knowledge, this is the first report on the use of a real-time PCR assay for identification of Brucella genotype ST27 in marine mammals.

Use of pooled protozoal cultures of preputial scraping samples obtained from bulls for the detection of Tritrichomonas foetus by means of a real-time polymerase chain reaction assay

To determine the sensitivity of a real-time PCR assay for the detection of Tritrichomonas foetus in protozoal cultures of preputial scraping samples pooled from up to 25 bulls and to determine the specificity of that assay for detection of T foetus in cultures for individual animals. Cross-sectional study. 188 bulls and 150 steers. Preputial scraping samples were collected, placed in a culture kit, and incubated at 37°C for 7 days. Cultures for individual animals were tested for T foetus by means of a real-time PCR assay. Pools of protozoal cultures were made by including fixed aliquots of samples with known positive and negative results in ratios of 1:2, 1:3, 1:5, 1:10, 1:15, 1:20, and 1:25. Specificities of the real-time PCR assay and culture for detection of T foetus in samples obtained from individual animals and sensitivity of real-time PCR assay for each evaluated pool ratio were determined. Specificity estimates for culture and the real-time PCR assay for detection of T foetus in preputial scraping samples for individual animals were not significantly different (98.8% and 100%, respectively). Sensitivities of the real-time PCR assay for the various pooled samples with known positive and negative T foetus results were not significantly different; overall sensitivity of the assay was 94%. Results indicated the evaluated real-time PCR assay had high specificity and good sensitivity for the detection of T foetus in pooled protozoal cultures of preputial scraping samples obtained from up to 25 animals.

Clinical Validation of the HPV-Risk Assay, a Novel Real-Time PCR Assay for Detection of High-Risk Human Papillomavirus DNA by Targeting the E7 Region

The HPV-Risk assay is a novel real-time PCR assay targeting the E7 region of 15 high-risk human papillomavirus (HPV) types (i.e., HPV16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -67, and -68), and provides additional genotype information for HPV16 and HPV18. This study evaluated the clinical performance and reproducibility of the HPV-Risk assay with cervical scraping specimens and its utility with self-collected (cervico)vaginal specimens. The clinical performance of the HPV-Risk assay for cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) with cervical scraping specimens was evaluated by a noninferiority analysis, relative to high-risk HPV GP5+/6+ PCR, following international guidelines for HPV test requirements for cervical cancer screening. The HPV-Risk assay showed clinical sensitivity for CIN2+ of 97.1% (95% confidence interval [CI], 89.1 to 99.3%; 67/69 samples) and a clinical specificity for CIN2+ of 94.3% (95% CI, 92.5 to 95.7%; 777/824 samples). The clinical sensitivity and specificity were noninferior to those of GP5+/6+ PCR (noninferiority score test, P=0.006 and 0.0003, respectively). Intralaboratory reproducibility over time (99.5% [95% CI, 98.6 to 99.8%]; 544/547 samples, kappa=0.99) and interlaboratory agreement (99.2% [95% CI, 98.6 to 99.8%]; 527/531 samples, kappa=0.98) for the HPV-Risk assay with cervical scraping specimens were high. The agreement of the HPV-Risk assay results for self-collected (cervico)vaginal specimens and clinician-obtained cervical scraping specimens was also high, i.e., 95.9% (95% CI, 85.1 to 99.0%; 47/49 samples, kappa=0.90) for self-collected lavage samples and 91.6% (95% CI, 84.6 to 95.6%; 98/107 samples, kappa=0.82) for self-collected brush samples. In conclusion, the HPV-Risk assay meets the cross-sectional clinical and reproducibility criteria of the international guidelines for HPV test requirements and can be considered clinically validated for cervical screening purposes. The compatibility of the HPV-Risk assay with self-collected specimens supports its utility for HPV self-sampling.

Development of a Duplex Real-Time PCR Method for the Pharmaceutical Rapid Microbial Detection of <i>Staphylococcus aureus</i> and <i>Pseudomonas aeruginosa</i>

Objective: To develop a duplex real-time PCR assay for pharmaceutical rapid microbial detection of Staphylococcus aureus and Pseudomonas aeruginosa. Methods: The specific primers and probes were designed to amplify the femB gene of S. aureus and the DNA gyrase subunit B gene of P. aeruginosa. The sensitivity of the system was detected by a multiple proportional dilution method. In order to examine the specificity of the system, other twenty-one bacteria strains were assayed simultaneously. Results: A highly sensitive and specific duplex real-time PCR assay for the detection of S. aureus and P. aeruginosa was established. The sensitivity was 50 copies/μL. The specificity was 100%. The whole detection procedure can be finished within 2.5 h. Conclusion: The duplex real-time PCR method is efficient in detecting with good sensitivity and specificity. There is a good prospect of this method applying in disease prevention and pharmaceutical industry due to the simultaneous detection of two pathogens.

Evaluation of Two Commercial Real-Time PCR Assays for Detection of Mycoplasma genitalium in Urogenital Specimens

The performance of two commercial real-time PCR kits for the detection of Mycoplasma genitalium was evaluated in comparison to an in-house real-time PCR assay. Concordances of 96% and 93% were found for the TIB MOLBIOL and the Diagenode assays, respectively, compared to the results of the in-house assay.

Evaluation of a Multitarget Real-Time PCR Assay for Detection of Bordetella Species during a Pertussis Outbreak in New Hampshire in 2011

A multitarget real-time PCR assay with three targets, including insertion sequence 481 (IS481), IS1001, and an IS1001-like element, as well as pertussis toxin subunit S1 (ptxS1), for the detection of Bordetella species was evaluated during a pertussis outbreak. The sensitivity and specificity were 77 and 88% (PCR) and 66 and 100% (culture), respectively. All patients with an IS481 C(T) of <30 also tested positive by ptxS1 assay and were clinical pertussis cases. No patients with IS481 C(T) values of ≥40 tested positive by culture. Therefore, we recommend that culture be performed only for specimens with IS481 C(T) values of 30 ≤ CT <40.

Presence of viral and bacterial organisms in milk and their association with somatic cell counts

About 20 to 35% of milk samples from cows with intramammary infection or high somatic cell count (SCC) are negative on bacteriological culture analysis. However, little is known about SCC in milk of cows infected with viruses. In the first part of our study, we developed a real-time PCR assay for detection of bovine herpesvirus (BHV) 1, BHV2, and BHV4, and bovine viral diarrhea virus (BVDV) in composite quarter milk samples. A total of 1,479 lactating cows of 1,964 cows in the dairy herd were initially selected because these cows had complete SCC data for at least 3 consecutive test results, of which 139 lactating cows from different lactation age groups were selected randomly and studied extensively. Composite quarter milk samples were collected on 3 alternate days and examined for viruses, SCC, and bacteriological analysis. In total, 10, 28, and 0.7% of the composite quarter milk samples from cows were positive for BHV1, BHV2, and BHV4, respectively; BVDV was not detected in composite quarter milk samples. Bovine herpesvirus was not associated with a particular bacterial species. Our study results indicate that cows positive for BHV in composite quarter milk samples alone are less likely to have elevated SCC compared with cows with bacterial intramammary infection; BHV1, BHV2, and BHV4 are probably not major udder pathogens.

Development and application of a real-time PCR assay for detection of Vibrio parahaemolyticuss in oyster

Vibrio parahaemolyticus(VP) is a pathogen that is the leading cause of shellfish-associated cases of bacterial food poisoning.To establish a real-time PCR for quantitative analysis of toxR gene,a pair of specific primers and TaqMan fluorescent probe of toxR gene of VP were designed.The positive recombinant plasmids of toxR gene served as templates to establish standard curve,which showed corresponding relationship between the copies of plasmids and threshold cycle(CT) values.The lowest limit detection was 15 copies of toxR gene for plasmid,and the sensitivity of pure cultures and simulated oyster sample was 18 CFU/mL and 180 CFU/g,respec-tively.The coefficient of variation was 0.95% for intra-assay test and 1.5% for inter-assay test.One hundred and sixty eight positively established samples were detected from 178 oysters collected from a farm in Guangdong Province.The result showed that the real-time PCR assay for VP had high specificity,sensitivity,repeatability and it was time saving,so it can be used for detecting and monitoring VP in aquatic products.

Development of a Taqman real-time PCR assay for detection of Leifsonia xyli subsp xyli

Leifsonia xyli subsp xyli (Lxx) is the etiological agent of ratoon-stunting disease (RSD), one of the most important diseases that affects productivity of sugarcane crops. The lack of defined external symptoms in most plants is a challenge for the diagnosis of RSD facilitating its spread in production fields. In this study a Taqman® real-time polymerase chain reaction (qPCR) assay was developed for detection of the bacteria in different tissues of the plants and compared to conventional PCR and nested-PCR assays. The bacterial DNA was amplified by the nested-PCR and qPCR assays, indicating that conventional PCR lacks sufficient sensitivity to be used as a diagnostic tool for RSD. On the other hand, qPCR is more advantageous than nested-PCR for processing large amounts of samples since it does not need post processing steps for visualization of results and the level of bacterial infection can be quantified. Therefore by improving the diagnosis of RSD in field plants, it can be applied to monitor losses caused by the disease, as a tool to monitor the efficacy of sanitation procedures and in the development of new sanitizers for Lxx.

A Newly Developed Real-Time PCR Assay for Detection and Quantification of Fusarium oxysporum and Its Use in Compatible and Incompatible Interactions with Grafted Melon Genotypes

A reliable and species-specific real-time quantitative polymerase chain reaction (qPCR) assay was developed for detection of the complex soilborne anamorphic fungus Fusarium oxysporum. The new primer pair, designed on the translation elongation factor 1-α gene with an amplicon of 142 bp, was highly specific to F. oxysporum without cross reactions with other Fusarium spp. The protocol was applied to grafted melon plants for the detection and quantification of F. oxysporum f. sp. melonis, a devastating pathogen of this cucurbit. Grafting technologies are widely used in melon to confer resistance against new virulent races of F. oxysporum f. sp. melonis, while maintaining the properties of valuable commercial varieties. However, the effects on the vascular pathogen colonization have not been fully investigated. Analyses were performed on 'Charentais-T' (susceptible) and 'Nad-1' (resistant) melon cultivars, both used either as rootstock and scion, and inoculated with F. oxysporum f. sp. melonis race 1 and race 1,2. Pathogen development was compared using qPCR and isolations from stem tissues. Early asymptomatic melon infections were detected with a quantification limit of 1 pg of fungal DNA. The qPCR protocol clearly showed that fungal development was highly affected by host-pathogen interaction (compatible or incompatible) and time (days postinoculation). The principal significant effect (P ≤ 0.01) on fungal development was due to the melon genotype used as rootstock, and this effect had a significant interaction with time and F. oxysporum f. sp. melonis race. In particular, the amount of race 1,2 DNA was significantly higher compared with that estimated for race 1 in the incompatible interaction at 18 days postinoculation. The two fungal races were always present in both the rootstock and scion of grafted plants in either the compatible or incompatible interaction.

One-step real-time PCR assay for detection and quantitation of hepatitis D virus RNA

Hepatitis D virus (HDV) is a defective virus which requires hepatitis B virus (HBV) surface antigen (HBsAg) for its assembly. Hepatitis B infected individuals co-infected or superinfected with HDV often present with more severe hepatitis, progress faster to liver disease, and have a higher mortality rate than individuals infected with HBV alone. Currently, there are no commercially available clinical tests for the detection and quantitation of HDV RNA in the United States. A one-step TaqMan quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for detection of HDV RNA, designing primers located in the region just downstream from the HDV antigen gene. The assay has the potential to detect all eight HDV genotypes. A quantifiable synthetic RNA control was also developed for use in the determination of HDV RNA titers in clinical samples. The limit of detection of this assay is 7.5×102 HDV RNA copies/ml with a dynamic range of six logs. Most clinical specimens tested (40/41) fell within the linear range of the assay. The median HDV RNA titer of the tested specimens was 6.24×106copies/ml, with a range of 8.52×103–1.79×109copies/ml. Out of 132 anti-HDV-positive specimens 41 (31.1%) were positive for HDV RNA.

P2.048 Evaluation of Two Commercial Real-Time PCR Assays For Detection of Mycoplasma Genitalium in Urogenital Specimens

ObjectivesMycoplama genitalium is a sexually transmitted organism associated with non-gonococcal urethritis in men and several inflammatory reproductive tract syndromes in women. Nucleic acid amplification tests are currently the only available...