Development of a Real-Time Reverse-Transcription PCR for Detection of Newcastle Disease Virus RNA in Clinical Samples

A real-time reverse transcriptase (RT)-PCR assay, applying light upon extension (LUX) fluorogenic primers, was developed for rapid and efficient detection of Newcastle disease virus (NDV). The method, which targets the fusion (F) protein gene of the viral genome, gave positive signal with all NDV isolates tested (32/32), while negative results were obtained with heterologous pathogens (35/35), including 13 avian influenza virus isolates. The detection limit of the assay was approximately 10+1.2 egg infectious dose (EID)50/0.2 ml and 10+2.2 EID50/0.2 ml for virus suspensions and spiked chicken fecal samples, respectively. As expressed in plasmid copy number, the procedure has a sensitivity of approximately 20 copies of the plasmid harboring the target gene. Due to its high specificity, sensitivity, and relative simplicity, the LUX RT-PCR assay provides a novel, rapid, and practical tool for the detection of NDV.

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Single-Nucleotide Polymorphism Analysis to Select Conserved Regions for an Improved Real-Time Reverse Transcription–PCR Test Specific for Newcastle Disease Virus

Avian Diseases ◽

10.1637/aviandiseases-d-19-00071 ◽

2019 ◽

Vol 63 (4) ◽

pp. 625

Author(s):

H. L. Ferreira ◽

D. L. Suarez

Keyword(s):

Single Nucleotide Polymorphism ◽

Newcastle Disease Virus ◽

Reverse Transcription ◽

Newcastle Disease ◽

Polymorphism Analysis ◽

Single Nucleotide Polymorphism Analysis ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Reverse Transcription Pcr ◽

Pcr Test

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Evaluation of a Real-Time Reverse Transcription-PCR Assay for Detection of Enterovirus D68 in Clinical Samples from an Outbreak in New York State in 2014

Journal of Clinical Microbiology ◽

10.1128/jcm.00358-15 ◽

2015 ◽

Vol 53 (6) ◽

pp. 1915-1920 ◽

Cited By ~ 19

Author(s):

Jian Zhuge ◽

Eric Vail ◽

Jeffrey L. Bush ◽

Lauren Singelakis ◽

Weihua Huang ◽

...

Keyword(s):

New York ◽

Real Time ◽

Reverse Transcription ◽

New York State ◽

Respiratory Illness ◽

Clinical Samples ◽

Sequencing Analysis ◽

York State ◽

Reverse Transcription Pcr ◽

Enterovirus D68

An outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68) infection was reported in mid-August 2014 in the United States. In this study, we evaluated the diagnostic utility of an EV-D68-specific real-time reverse transcription-PCR (rRT-PCR) that was recently developed by the Centers for Disease Control and Prevention in clinical samples. Nasopharyngeal (NP) swab specimens from patients in a recent outbreak of respiratory illness in the lower Hudson Valley, New York State, were collected and examined for the presence of human rhinovirus or enterovirus using the FilmArray Respiratory Panel (RP) assay. Samples positive by RP were assessed using EV-D68 rRT-PCR, and the data were compared to results from sequencing analysis of partial VP1 and 5′ untranslated region (5′-UTR) sequences of the EV genome. A total of 285 RP-positive NP specimens (260 from the 2014 outbreak and 25 from 2013) were analyzed by rRT-PCR; EV-D68 was detected in 74 of 285 (26.0%) specimens examined. Data for comparisons between rRT-PCR and sequencing analysis were obtained from 194 NP specimens. EV-D68 detection was confirmed by sequencing analysis in 71 of 74 positive and in 1 of 120 randomly selected negative specimens by rRT-PCR. The EV-D68 rRT-PCR showed diagnostic sensitivity and specificity of 98.6% and 97.5%, respectively. Our data suggest that the EV-D68 rRT-PCR is a reliable assay for detection of EV-D68 in clinical samples and has a potential to be used as a tool for rapid diagnosis and outbreak investigation of EV-D68-associated infections in clinical and public health laboratories.

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