Abundance of Dioxygenase Genes Similar to Ralstonia sp. Strain U2 nagAc Is Correlated with Naphthalene Concentrations in Coal Tar-Contaminated Freshwater Sediments

ABSTRACT We designed a real-time PCR assay able to recognize dioxygenase large-subunit gene sequences with more than 90% similarity to the Ralstonia sp. strain U2 nagAc gene (nagAc-like gene sequences) in order to study the importance of organisms carrying these genes in the biodegradation of naphthalene. Sequencing of PCR products indicated that this real-time PCR assay was specific and able to detect a variety of nagAc-like gene sequences. One to 100 ng of contaminated-sediment total DNA in 25-μl reaction mixtures produced an amplification efficiency of 0.97 without evident PCR inhibition. The assay was applied to surficial freshwater sediment samples obtained in or in close proximity to a coal tar-contaminated Superfund site. Naphthalene concentrations in the analyzed samples varied between 0.18 and 106 mg/kg of dry weight sediment. The assay for nagAc-like sequences indicated the presence of (4.1 ± 0.7) × 103 to (2.9 ± 0.3) × 105 copies of nagAc-like dioxygenase genes per μg of DNA extracted from sediment samples. These values corresponded to (1.2 ± 0.6) × 105 to (5.4 ± 0.4) × 107 copies of this target per g of dry weight sediment when losses of DNA during extraction were taken into account. There was a positive correlation between naphthalene concentrations and nagAc-like gene copies per microgram of DNA (r = 0.89) and per gram of dry weight sediment (r = 0.77). These results provide evidence of the ecological significance of organisms carrying nagAc-like genes in the biodegradation of naphthalene.

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Real-time quantitative PCR for enteric adenovirus serotype 40 in environmental waters

Canadian Journal of Microbiology ◽

10.1139/w05-016 ◽

2005 ◽

Vol 51 (5) ◽

pp. 393-398 ◽

Cited By ~ 31

Author(s):

Sunny Jiang ◽

Hojabr Dezfulian ◽

Weiping Chu

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Amplification Efficiency ◽

Sewage Effluent ◽

Hexon Gene ◽

Pcr Assay ◽

Environmental Waters ◽

Cycle Number ◽

The Real

Adenoviruses 40 and 41 have been recognized as important etiological agents of gastroenteritis in children. A real-time PCR method (TaqMan® assay) was developed for rapid quantification of adenovirus 40 (Ad40) by amplifying an 88 bp sequence from the hexon gene. To establish a quantification standard curve, a 1090 bp hexon region of Ad40 was amplified and cloned into the pGEM®-T Vector. A direct correlation was observed between the fluorescence threshold cycle number (Ct) and the starting quantity of Ad40 hexon gene. The quantification was linear over 6-log units and the amplification efficiency averaged greater than 95%. Seeding studies using various environmental matrices (including sterile water, creek water, brackish estuarine water, ocean water, and secondary sewage effluent) suggest that this method is applicable to environmental samples. However, real-time PCR was sensitive to inhibitors present in the environmental samples. Lower efficiency of PCR amplification was found in secondary sewage effluent and creek waters. Application of the method to fecal contaminated waters successfully quantified the presence of Ad40. The sensitivity of the real-time PCR is comparable to the traditional nested PCR assay for environmental samples. In addition, the real-time PCR assay offers the advantage of speed and insensitivity to contamination during PCR set up. The real-time PCR assay developed in this study is suitable for quantitative determination of Ad40 in environmental samples and represents a considerable advancement in pathogen quantification in aquatic environments.Key words: adenovirus, real-time PCR, environmental waters, serotype 40.

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Development and validation of a highly sensitive real-time PCR assay for rapid detection of parapoxviruses

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638716680676 ◽

2016 ◽

Vol 29 (4) ◽

pp. 499-507 ◽

Cited By ~ 2

Author(s):

Amaresh Das ◽

Gordon Ward ◽

Andre Lowe ◽

Lizhe Xu ◽

Karen Moran ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Positive Control ◽

Nucleotide Sequencing ◽

Amplification Efficiency ◽

Analytical Sensitivity ◽

Pcr Assay ◽

Highly Sensitive ◽

Development And Validation

Parapoxviruses (PaPVs) cause widespread infections in ruminants worldwide. All PaPVs are zoonotic and may infect humans after direct or indirect contact with infected animals. Herein we report the development and validation of a highly sensitive real-time PCR assay for rapid detection of PaPVs. The new assay (referred to as the RVSS assay) was specific for PaPVs only and had no cross-reactivity against other pox viruses. Using a recombinant plasmid as positive control, the analytical sensitivity of the assay was determined to be 16 genome copies of PaPV per assay. The amplification efficiency estimate (91–99%), the intra- and interassay variability estimate (standard deviation [SD]: 0.28–1.06 and 0.01–0.14, respectively), and the operator variability estimate (SD: 0.78 between laboratories and 0.28 between operators within a laboratory) were within the acceptable range. The diagnostic specificity was assessed on 100 specimens from healthy normal animals and all but 1 tested negative (99%). The diagnostic sensitivity (DSe) was assessed on 77 clinical specimens (skin/scab) from infected sheep, goats, and cattle, and all tested positive (100%). The assay was multiplexed with beta-actin as an internal positive control, and the multiplex assay exhibited the same DSe as the singleplex assay. Further characterization of the PaPV specimens by species-specific real-time PCR and nucleotide sequencing of the PCR products following conventional PCR showed the presence of Orf virus not only in sheep and goats but also in 1 bovid. The validated RVSS assay demonstrated high specificity, sensitivity, reproducibility, and ruggedness, which are critical for laboratory detection of PaPVs.

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Development of a two-step nucleic acid amplification test for accurate diagnosis of the Mycobacterium tuberculosis complex

Scientific Reports ◽

10.1038/s41598-021-85160-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Chien-Ru Lin ◽

Hsin-Yao Wang ◽

Ting-Wei Lin ◽

Jang-Jih Lu ◽

Jason Chia-Hsun Hsieh ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Nucleic Acid ◽

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Disease Transmission ◽

High Sensitivity ◽

Nucleic Acid Amplification ◽

Amplification Efficiency ◽

Pcr Assay

AbstractThe Mycobacterium tuberculosis complex (MTBC) remains one of the top 10 leading causes of death globally. The early diagnosis of MTBC can reduce mortality and mitigate disease transmission. However, current nucleic acid amplification diagnostic test methods are generally time-consuming and show suboptimal diagnostic performance, especially in extrapulmonary MTBC samples or acid-fast stain (AFS)-negative cases. Thus, development of an accurate assay for the diagnosis of MTBC is necessary, particularly under the above mentioned conditions. In this study, a single-tube nested real-time PCR assay (N-RTP) was developed and compared with a newly in-house-developed high-sensitivity real-time PCR assay (HS-RTP) using 134 clinical specimens (including 73 pulmonary and 61 extrapulmonary specimens). The amplification efficiency of HS-RTP and N-RTP was 99.8% and 100.7%, respectively. The sensitivity and specificity of HS-RTP and N-RTP for the diagnosis of MTBC in these specimens were 97.5% (77/79) versus 94.9% (75/79) and 80.0% (44/55) versus 89.1% (49/55), respectively. The sensitivity and specificity of HS-RTP and N-RTP for the diagnosis of MTBC in pulmonary specimens were 96.3% (52/54) versus 96.3% (52/54) and 73.7.0% (14/19) versus 89.5% (17/19), respectively; in extrapulmonary specimens, the sensitivity and specificity of HS-RTP and N-RTP were 100% (25/25) versus 92% (23/25) and 83.3% (30/36) versus 88.9% (32/36), respectively. Among the AFS-negative cases, the sensitivity and specificity of HS-RTP and N-RTP were 97.0% (32/33) versus 90.9% (30/33) and 88.0% (44/50) versus 92.0% (46/50), respectively. Overall, the sensitivity of HS-RTP was higher than that of N-RTP, and the performance was not compromised in extrapulmonary specimens and under AFS-negative conditions. In contrast, the specificity of the N-RTP assay was higher than that of the HS-RTP assay in all types of specimens. In conclusion, the HS-RTP assay would be useful for screening patients suspected of exhibiting an MTBC infection due to its higher sensitivity, while the N-RTP assay could be used for confirmation because of its higher specificity. Our results provide a two-step method (screen to confirm) that simultaneously achieves high sensitivity and specificity in the diagnosis of MTBC.

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A Real-Time PCR Assay for Detection of Low Pneumocystis jirovecii Levels

Frontiers in Microbiology ◽

10.3389/fmicb.2021.787554 ◽

2022 ◽

Vol 12 ◽

Author(s):

Susana Ruiz-Ruiz ◽

Carolina A. Ponce ◽

Nicole Pesantes ◽

Rebeca Bustamante ◽

Gianna Gatti ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Nested Pcr ◽

Large Subunit ◽

Pcr Amplification ◽

Surface Protein ◽

Pneumocystis Jirovecii ◽

Pcr Assay ◽

Low Levels ◽

Large Subunit Rrna

Here we report a new real-time PCR assay using SYBR Green which provides higher sensitivity for the specific detection of low levels of Pneumocystis jirovecii. To do so, two primer sets were designed, targeting the family of genes that code for the most abundant surface protein of Pneumocystis spp., namely the major surface glycoproteins (Msg), and the mitochondrial large subunit rRNA (mtLSUrRNA) multicopy gene, simultaneously detecting two regions. PCR methods are instrumental in detecting these low levels; however, current nested-PCR methods are time-consuming and complex. To validate our new real-time Msg-A/mtLSUrRNA PCR protocol, we compared it with nested-PCR based on the detection of Pneumocystis mitochondrial large subunit rRNA (mtLSUrRNA), one of the main targets used to detect this pathogen. All samples identified as positive by the nested-PCR method were found positive using our new real-time PCR protocol, which also detected P. jirovecii in three nasal aspirate samples that were negative for both rounds of nested-PCR. Furthermore, we read both rounds of the nested-PCR results for comparison and found that some samples with no PCR amplification, or with a feeble band in the first round, correlated with higher Ct values in our real-time Msg-A/mtLSUrRNA PCR. This finding demonstrates the ability of this new single-round protocol to detect low Pneumocystis levels. This new assay provides a valuable alternative for P. jirovecii detection, as it is both rapid and sensitive.

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Real-Time PCR Assay for the Detection and Quantification of Roe Deer to Detect Food Adulteration—Interlaboratory Validation Involving Laboratories in Austria, Germany, and Switzerland

Foods ◽

10.3390/foods10112645 ◽

2021 ◽

Vol 10 (11) ◽

pp. 2645

Author(s):

Barbara Druml ◽

Steffen Uhlig ◽

Kirsten Simon ◽

Kirstin Frost ◽

Karina Hettwer ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Roe Deer ◽

Amplification Efficiency ◽

Pcr Assay ◽

Food Adulteration ◽

The Real ◽

Game Meat ◽

Meat Samples ◽

Detection And Quantification

Game meat products are particularly prone to be adulterated by replacing game meat with cheaper meat species. Recently, we have presented a real-time polymerase chain reaction (PCR) assay for the identification and quantification of roe deer in food. Quantification of the roe deer content in % (w/w) was achieved relatively by subjecting the DNA isolates to a reference real-time PCR assay in addition to the real-time PCR assay for roe deer. Aiming at harmonizing analytical methods for food authentication across EU Member States, the real-time PCR assay for roe deer has been tested in an interlaboratory ring trial including 14 laboratories from Austria, Germany, and Switzerland. Participating laboratories obtained aliquots of DNA isolates from a meat mixture containing 24.8% (w/w) roe deer in pork, roe deer meat, and 12 meat samples whose roe deer content was not disclosed. Performance characteristics included amplification efficiency, level of detection (LOD95%), repeatability, reproducibility, and accuracy of quantitative results. With a relative reproducibility standard deviation ranging from 13.35 to 25.08% (after outlier removal) and recoveries ranging from 84.4 to 114.3%, the real-time PCR assay was found to be applicable for the detection and quantification of roe deer in raw meat samples to detect food adulteration.

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Development of a Rapid and Fully Automated Multiplex Real-Time PCR Assay for Identification and Differentiation of Vibrio cholerae and Vibrio parahaemolyticus on the BD MAX Platform

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.639473 ◽

2021 ◽

Vol 11 ◽

Author(s):

Zhenpeng Li ◽

Hongxia Guan ◽

Wei Wang ◽

He Gao ◽

Weihong Feng ◽

...

Keyword(s):

Vibrio Cholerae ◽

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus ◽

Health Concern ◽

Specific Gene ◽

Amplification Efficiency ◽

Pcr Assay ◽

Percent Agreement ◽

Stool Samples

Vibrio cholerae and Vibrio parahaemolyticus are common diarrheal pathogens of great public health concern. A multiplex TaqMan-based real-time PCR assay was developed on the BD MAX platform; this assay can simultaneously detect and differentiate V. cholerae and V. parahaemolyticus directly from human fecal specimens. The assay includes two reactions. One reaction, BDM-VC, targets the gene ompW, the cholera toxin (CT) coding gene ctxA, the O1 serogroup specific gene rfbN, and the O139 serogroup specific gene wbfR of V. cholerae. The other, BDM-VP, targets the gene toxR and the toxin coding genes tdh and trh of V. parahaemolyticus. In addition, each reaction contains a sample process control. When evaluated with spiked stool samples, the detection limit of the BD MAX assay was 195–780 CFU/ml for V. cholerae and 46–184 CFU/ml for V. parahaemolyticus, and the amplification efficiency of all genes was between 95 and 115%. The assay showed 100% analytical specificity, using 63 isolates. The BD MAX assay was evaluated for its performance compared with conventional real-time PCR after automated DNA extraction steps, using 164 retrospective stool samples. The overall percent agreement between the BD MAX assay and real-time PCR was ≥ 98.8%; the positive percent agreement was 85.7% for ompW, 100% for toxR/tdh, and lower (66.7%) for trh because of a false negative. This is the first report to evaluate the usage of the BD MAX open system in detection and differentiation of V. cholerae and V. parahaemolyticus directly from human samples.

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Investigation of the Food-Transmitted Parasites Trichinella spp. and Alaria spp. in Wild Boars in Greece by Classical and Molecular Methods and Development of a Novel Real-Time PCR for Alaria spp. Detection

Animals ◽

10.3390/ani11102803 ◽

2021 ◽

Vol 11 (10) ◽

pp. 2803

Author(s):

Dimitris Dimzas ◽

Taxiarchis Chassalevris ◽

Zanda Ozolina ◽

Chrysostomos I. Dovas ◽

Anastasia Diakou

Keyword(s):

Wild Boar ◽

Real Time ◽

Real Time Pcr ◽

Large Subunit ◽

Taqman Probe ◽

Amplification Efficiency ◽

Wild Boars ◽

The Usa ◽

Wild Boar Meat ◽

Low Prevalence

Foodborne parasitic diseases represent a major threat to public health. Trichinellosis, caused by the nematode parasite Trichinella spp., is one of the most important foodborne diseases, while alariosis, caused by the trematode parasite Alaria spp., is less common in humans, and rare cases have been reported only in the USA and Canada. Both parasites can infect humans via the consumption of raw or undercooked wild boar meat. In order to investigate the prevalence of these parasites in wild boar meat in Greece, samples from the diaphragm pillars and the region of the mandibular angle from 128 wild boars, hunted in Greece, were collected. The samples were examined by classical parasitological (compression, artificial digestion, and Alaria spp. migration) and by molecular (real-time PCR) methods. For Trichinella spp. an existent real-time PCR detecting all species likely to be present in Greece was applied, while for Alaria spp. a real-time PCR was developed, employing an LNA TaqMan probe targeting the large subunit ribosomal RNA gene. All examined wild boar samples from Greece resulted negative for Trichinella and Alaria species, indicating a low prevalence of infection in the examined population. The novel real-time PCR for Alaria spp. has 81.5% amplification efficiency and is able to detect 0.12 larvae per 50 g of tissue and could be utilized as a complementary to AMT diagnostic tool in surveillance.

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Triplex Real-Time PCR Approach for the Detection of Crucial Fungal Berry Pathogens—Botrytis spp., Colletotrichum spp. and Verticillium spp.

International Journal of Molecular Sciences ◽

10.3390/ijms21228469 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8469

Author(s):

Dominika G. Malarczyk ◽

Jacek Panek ◽

Magdalena Frąc

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Production Efficiency ◽

Detection Method ◽

Large Subunit ◽

Main Concern ◽

Pcr Assay ◽

Protective Measures ◽

Fluorogenic Probes ◽

Colletotrichum Spp

Phytopathogens cause undeniably serious damage in agriculture by harming fruit cultivations and lowering harvest yields, which as a consequence substantially reduces food production efficiency. Fungi of the Botrytis, Colletotrichum and Verticillium genera are a main concern in berry production. However, no rapid detection method for detecting all of these pathogens simultaneously has been developed to date. Therefore, in this study, a multiplex real-time PCR assay for this purpose was established. Universal fungal primers for the D2 region of the large subunit ribosomal DNA and three multiplexable fluorogenic probes specific for the chosen fungi were designed and deployed. The triplex approach for the molecular detection of these fungi, which was developed in this study, allows for the rapid and effective detection of crucial berry pathogens, which contributes to a more rapid implementation of protective measures in plantations and a significant reduction in losses caused by fungal diseases.

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