Detection of Mycoplasma hyopneumoniae by Air Sampling with a Nested PCR Assay

1998 ◽  
Vol 64 (2) ◽  
pp. 543-548 ◽  
Author(s):  
Katharina D. C. Stärk ◽  
Jacques Nicolet ◽  
Joachim Frey
Keyword(s):  
Nested Pcr ◽  
Gene Segment ◽  
Mycoplasma Hyopneumoniae ◽  
Field Conditions ◽  
Pcr Analysis ◽  
Direct Extraction ◽  
Pcr Assay ◽  
Sampling System ◽  
Respiratory Problems ◽  
One Step

ABSTRACT This article describes the first successful detection of airborneMycoplasma hyopneumoniae under experimental and field conditions with a new nested PCR assay. Air was sampled with polyethersulfone membranes (pore size, 0.2 μm) mounted in filter holders. Filters were processed by dissolution and direct extraction of DNA for PCR analysis. For the PCR, two nested pairs of oligonucleotide primers were designed by using an M. hyopneumoniae-specific DNA sequence of a repeated gene segment. A nested PCR assay was developed and used to analyze samples collected in eight pig houses where respiratory problems had been common. Air was also sampled from a mycoplasma-free herd. The nested PCR was highly specific and 104 times as sensitive as a one-step PCR. Under field conditions, the sampling system was able to detect airborne M. hyopneumoniae on 80% of farms where acute respiratory disease was present. No airborne M. hyopneumoniae was detected on infected farms without acute cases. The chance of successful detection was increased if air was sampled at several locations within a room and at a lower air humidity.

scholarly journals Detection of Phytoplasmas in Declining Pears in Southern Australia

Plant Disease ◽  
1997 ◽  
Vol 81 (3) ◽  
pp. 254-258 ◽  
Author(s):  
B. Schneider ◽  
K. S. Gibb
Keyword(s):  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Analysis ◽  
Pcr Assay ◽  
Specific Primers ◽  
Pear Tree ◽  
Pear Trees ◽  
Polymerase Chain

Forty-nine pear tree samples collected in Victoria, most of them showing decline symptoms, were tested by polymerase chain reaction (PCR) analysis to detect phytoplasmas. Two universal phytoplasma-specific primer pairs, fP1/rP7 and fU5/rU3, were tested, but only fU5/rU3 amplified the phytoplasma DNA adequately. Nested PCR with universal and group-specific primers, however, proved more effective. Thirty pear trees reacted positively in a nested PCR assay. Restriction fragment length polymorphism (RFLP) analysis with the restriction enzymes MseI and AluI of the PCR fragment amplified with the primer pair fU5/rU3 revealed patterns identical to those from the sweet potato little leaf phytoplasma. This is the first report of a phytoplasma in pear in Australia.

A nested PCR assay for the detection of Mycoplasma hyopneumoniae in tracheobronchiolar washings from pigs

2000 ◽  
Vol 76 (1) ◽  
pp. 31-40 ◽  
Author(s):  
E Verdin ◽  
C Saillard ◽  
A Labbé ◽  
J.M Bové ◽  
M Kobisch
Keyword(s):  
Nested Pcr ◽  
Mycoplasma Hyopneumoniae ◽  
Pcr Assay

scholarly journals A Nested PCR Assay for Detecting Valsa mali var. mali in Different Tissues of Apple Trees

Plant Disease ◽  
2012 ◽  
Vol 96 (11) ◽  
pp. 1645-1652 ◽  
Author(s):  
Rui Zang ◽  
Zhiyuan Yin ◽  
Xiwang Ke ◽  
Xiaojie Wang ◽  
Zhengli Li ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Detection Rate ◽  
Pcr Analysis ◽  
Pcr Assay ◽  
Apple Trees ◽  
Specific Primers ◽  
Fungal Isolation ◽  
Species Specific ◽  
Valsa Mali ◽  
Different Tissues

A nested polymerase chain reaction (PCR) assay for detecting Valsa mali var. mali, the causal agent of apple tree Valsa canker, was developed. One pair of genus-specific primers was designed based on the ribosomal DNA internal transcribed spacer conservative sequence of the Valsa genus and one pair of species-specific primers was designed based on the specific sequence of V. mali var. mali. The specificity of the genus-specific and species-specific primers was evaluated against 10 V. mali var. mali isolates, 10 V. mali var. pyri isolates, 4 isolates from closely related Valsa spp., and 8 isolates from fungal species that are commonly isolated from naturally infected apple bark tissue. A distinct band of 348 bp in length was detected in all V. mali var. mali isolates but not in other tested species and the V. mali var. pyri variety. The sensitivity of this assay was evaluated by serial dilutions of DNA extracted from V. mali var. mali pure cultures and apple bark tissues with or without visible symptoms. The results showed that the assay was able to detect as little as 100 fg of DNA in mycelial samples and apple bark tissues with visible symptoms, whereas the lowest detectable concentration was 10 pg of DNA in symptomless apple bark tissues. The efficiency of the nested PCR assay was compared with that of fungal isolation assays. All symptomless and symptomatic samples from which the pathogen was successfully isolated yielded a PCR product of the expected size. The detection rate of nested PCR for symptomless samples was 64.7%, which was much higher than the detection rate of 20.6% by fungal isolation. The PCR analysis of different symptomless tissues showed that the incidence of V. mali var. mali was different in different tissues of apple trees. The average incidence of V. mali var. mali was 89% in terminal buds, 71% in internodes, and 48% in bud scale scars. Moreover, the incidence of V. mali var. mali in nonsymptomatic tissues was higher in orchards where more trees were infected. Taken together, the assay developed in this study can be used for rapid and reliable detection of V. mali var. mali in tissues of apple trees with or without symptoms and also for monitoring the presence of the pathogen at an early stage of disease development.

scholarly journals Comprehensive and Rapid Real-Time PCR Analysis of 21 Foodborne Outbreaks

2009 ◽  
Vol 2009 ◽  
pp. 1-13 ◽  
Author(s):  
Hiroshi Fukushima ◽  
Kazunori Katsube ◽  
Yoshie Tsunomori ◽  
Ryoko Kishi ◽  
Junko Atsuta ◽  
...  
Keyword(s):  
Foodborne Pathogens ◽  
Real Time Pcr ◽  
Pcr Analysis ◽  
Pcr Assay ◽  
Causative Agents ◽  
Foodborne Outbreaks ◽  
One Step ◽  
Stool Specimens ◽  
Effective Diagnosis ◽  
Almost All

A set of four duplex SYBR Green I PCR (SG-PCR) assay combined with DNA extraction using QIAamp DNA Stool Mini kit was evaluated for the detection of foodborne bacteria from 21 foodborne outbreaks. The causative pathogens were detected in almost all cases in 2 hours or less. The first run was for the detection of 8 main foodborne pathogens in 5 stool specimens within 2 hours and the second run was for the detection of other unusual suspect pathogens within a further 45 minutes. After 2 to 4 days, the causative agents were isolated and identified. The results proved that for comprehensive and rapid molecular diagnosis in foodborne outbreaks, Duplex SG-PCR assay is not only very useful, but is also economically viable for one-step differentiation of causative pathogens in fecal specimens obtained from symptomatic patients. This then allows for effective diagnosis and management of foodborne outbreaks.

A duplex nested-PCR assay for detection of mud crab reovirus and mud crab dicistrovirus-1

2013 ◽  
Vol 20 (4) ◽  
pp. 808-815
Author(s):  
Di ZHANG ◽  
Keng YANG ◽  
Youlu SU ◽  
Juan FENG ◽  
Zhixun GUO
Keyword(s):  
Nested Pcr ◽  
Mud Crab ◽  
Pcr Assay

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV

2013 ◽  
Vol 189 (2) ◽  
pp. 277-282 ◽  
Author(s):  
Yong Yan ◽  
Heng-hui Wang ◽  
Lei Gao ◽  
Ji-mei Ji ◽  
Zhi-jie Ge ◽  
...  
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Human Norovirus ◽  
One Step
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