In Situ Hybridization for the Detection and Localization of Porcine Epidemic Diarrhea Virus in the Intestinal Tissues from Naturally Infected Piglets

Kim, O.; Chae, Chanhee

doi:10.1354/vp.37-1-62

Cited by 50 publications

(41 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Even now, the viral titer of PEDV in cell culture is still low. Other diagnostic methods for detecting PEDV include immunohistochemistry, in situ hybridization, dot-blot hybridization, RT-PCR, and real-time RT-PCR [24][25][26][27][28]. These methods may require either high-precision instruments or complicated procedures.…”

Section: Discussionmentioning

confidence: 99%

Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus

Ren

2011

Virus Genes

View full text Add to dashboard Cite

In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for detection of porcine epidemic diarrhea virus (PEDV). Six primers were designed to amplify the nucleocapsid (N) gene of PEDV. The optimization, sensitivity, and specificity of the RT-LAMP were investigated. The results showed that the optimal reaction condition for RT-LAMP amplifying PEDV N gene was achieved at 63°C for 50 min. The RT-LAMP assay was more sensitive than gel-based RT-PCR and enzyme-linked immunosorbent assay. It was capable of detecting PEDV from clinical samples and differentiating PEDV from Porcine transmissible gastroenteritis virus, Porcine rotavirus, Porcine pseudorabies virus, Porcine reproductive and respiratory syndrome virus, and Avian infectious bronchitis virus.

show abstract

Section: Discussionmentioning

confidence: 99%

Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus

Ren

2011

Virus Genes

View full text Add to dashboard Cite

show abstract

“…In situ hybridization techniques have been used successfully to detect viral nucleic acids with DNA probes complementary to viral RNA. 7,16 One objective of this study was to detect SIV nucleic acids in formalin-fixed, paraffin-embedded tissues using a digoxigenin-labeled 582-base pair (bp) single-stranded DNA probe complementary to a conserved region of the SIV nucleocapsid protein gene. The second objective was to determine the location of SIV replication in the porcine lung to better understand the pathogenesis of naturally occurring SIV infection.…”

mentioning

confidence: 99%

Localization of Swine Influenza Virus in Naturally Infected Pigs

2002

Self Cite

View full text Add to dashboard Cite

Abstract. Swine influenza virus (SIV) RNA and antigen were detected in 15 naturally infected pigs by in situ hybridization using a nonradioactive digoxigenin-labeled cDNA probe and by immunohistochemistry using an influenza virus H1N1-specific monoclonal antibody. A 582-base pair cDNA probe for viral RNA encoding the nucleocapsid protein of SIV type A H1N1 strain was generated by the reverse transcription polymerase chain reaction. In situ hybridization and immunohistochemistry gave similar results for serial sections from each of 15 lung samples. Positive cells typically exhibited a dark brown (in situ hybridization) or red (immunohistochemistry) reaction product in the nucleus and cytoplasm without background staining. A strong positive signal for both in situ hybridization and immunohistochemistry was detected mainly in the bronchial and bronchiolar epithelial cells. A less intense signal was detected in the interstitial and alveolar macrophages. Simultaneous detection of hybridization and immunohistochemical signals on serial sections provided evidence that SIV had replicated in positive cells. The in situ hybridization technique developed in this study was useful for the detection of SIV RNA in tissues taken from naturally infected pigs and may be a valuable technique for studying the pathogenesis of SIV infection.

show abstract

“…The PCR products were purified with Wizard PCR Preps (Promega Biotech, Madison, WI, U.S.A.) and labeled by random priming with digoxigenin-dUTP with a commercial kit (Boehringer Mannheim, Indianapolis, IN, U.S.A.) according to the manufacturer's instructions. ISH was carried out as described previously [5].…”

Section: Preparation Of Infected Cellsmentioning

confidence: 99%

Development of in situ Nest PCR and Comparison of Five Molecular Biological Diagnostic Methods for the Detection of Intracellular Viral DNAs in Paraffin Sections.

Kim

2003

The Journal of Veterinary Medical Science

View full text Add to dashboard Cite

ABSTRACT. Nest polymerase chain reaction (PCR), in situ hybridization (ISH), in situ PCR, in situ PCR/hybridization (PCR-ISH) and in situ nest PCR were compared for the detection and localization of intracellular viral DNAs in paraffin sections. MDBK cells were infected with alcelapine herpesvirus 1 ranging from 10 1 to 10 5 50% tissue culture infected doses (TCID 50 ), incubated 18 hr, then fixed and processed into paraffin blocks. Sections of the cell preparation were subjected to nest PCR, ISH, in situ PCR, PCR-ISH and in situ nest PCR using specific oligonucleotide primers or probes directed against the viral open reading frame 50. In situ nest PCR and nest PCR were found to be capable of detecting the viral DNA in the cells infected with the lowest virus titer. As compared with other molecular biological methods for the detection of the virus, in situ nest PCR was found to be more sensitive than ISH, in situ PCR and PCR-ISH. In situ nest PCR has wide applications for sensitive localization of low copy viral sequences within cells to investigate the role of viruses in a variety of clinical conditions. KEY WORDS: in situ hybridization, in situ PCR, in situ PCR/hybridization, in situ nest PCR.

show abstract

In Situ Hybridization for the Detection and Localization of Porcine Epidemic Diarrhea Virus in the Intestinal Tissues from Naturally Infected Piglets

Cited by 50 publications

References 24 publications

Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus

Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus

Localization of Swine Influenza Virus in Naturally Infected Pigs

Development of in situ Nest PCR and Comparison of Five Molecular Biological Diagnostic Methods for the Detection of Intracellular Viral DNAs in Paraffin Sections.

Contact Info

Product

Resources

About