A semi-automated and highly sensitive streptavidin magnetic capture-hybridization RT-PCR assay: Application to genus-wide or species-specific detection of several viruses of ornamental bulb crops

This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5′ noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.

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Development of a nanoparticle-assisted PCR assay to distinguish canine coronaviruses I and II

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638720974114 ◽

2020 ◽

Vol 33 (1) ◽

pp. 104-107

Author(s):

Tong Qin ◽

Jing Wang ◽

Shang-Jin Cui

Keyword(s):

Mixed Infection ◽

Annealing Temperature ◽

Clinical Samples ◽

Rt Pcr ◽

M Gene ◽

Specific Detection ◽

Pcr Assay ◽

Reaction Conditions ◽

Novel Method ◽

Beijing China

Nanoparticle-assisted PCR (nanoPCR) is a novel method for the simple, rapid, and specific detection of viruses. We developed a nanoPCR method to detect and differentiate canine coronavirus I (CCoV I) and II (CCoV II). Primer pairs were designed against the M gene conserved region of CCoV I and CCoV II, producing specific fragments of 239 bp (CCoV I) and 105 bp (CCoV II). We optimized the annealing temperature and primer concentrations for the CCoV nanoPCR assay and assessed its sensitivity and specificity. Under optimized nanoPCR reaction conditions, the detection limits were 6.47 × 101 copies/μL for CCoV I and 6.91 × 102 copies/μL for CCoV II. No fragments were amplified using other canine viruses as templates. The sensitivity of the nanoPCR assay was 100-fold higher than that of a conventional RT-PCR assay. Among 60 clinical samples collected from Beijing, China, the assay detected 12% positive for CCoV I and 48% positive for CCoV II. Our nanoPCR method is an effective method to rapidly detect CCoV I and CCoV II alone, or as a mixed infection, in dogs.

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Identification of donkey meat in foods using species-specific PCR combined with lateral flow immunoassay

RSC Advances ◽

10.1039/c9ra05060d ◽

2019 ◽

Vol 9 (46) ◽

pp. 26552-26558 ◽

Cited By ~ 1

Author(s):

Liangjuan Zhao ◽

Marti Z. Hua ◽

Shenmiao Li ◽

Jinyu Liu ◽

Wenjie Zheng ◽

...

Keyword(s):

Low Cost ◽

Lateral Flow ◽

Lateral Flow Immunoassay ◽

Specific Detection ◽

Chain Reaction ◽

Specific Pcr ◽

Highly Sensitive ◽

Polymerase Chain ◽

Species Specific ◽

Government Laboratories

The developed species-specific polymerase chain reaction-lateral flow immunoassay (PCR-LFI) method allows the rapid, low-cost, highly sensitive and specific detection of donkey DNA for meat authentication, adopted by government laboratories.

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Multiplex TaqMan Real-Time PCR Assay for Sensitive Detection of Two Weevil Species (Coleoptera: Curculionidae)

Journal of Economic Entomology ◽

10.1093/jee/toaa251 ◽

2020 ◽

Author(s):

Carlos Aguirre ◽

Evelyn Sánchez ◽

Natalia Olivares ◽

Patricio Hinrichsen

Keyword(s):

Real Time ◽

Citrus Fruit ◽

Sensitive Detection ◽

Morphological Identification ◽

Specific Detection ◽

Pcr Assay ◽

Polymerase Chain ◽

Cost Efficient ◽

Species Specific ◽

Template Dna

Abstract Rapid and cost-efficient identification of Naupactus species is becoming a key process for the exportation of citrus fruit from Chile and other countries, considering the quarantine regulations for some species of the cosmopolitan genus Naupactus. This study deals with the development of a fast and sensitive detection protocol for Naupactus cervinus (Coleoptera: Curculionidae) (Boheman) and Naupactus xanthographus (Coleoptera: Curculionidae) (Germar) based on multiplex TaqMan Real-time polymerase chain reaction. Both N. cervinus and N. xanthographus primer and probe sets achieved species-specific detection in a linear range from 1 pg/μl to 1 × 10-6 pg/μl, allowing detection of as few as 160 copies of template DNA. Non-target amplifications were not detected and a panel composed of 480 test samples had 100% coincidence with the respective morphological identification.

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