Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Takekawa, John Y.; Hill, Nichola J.; Schultz, Annie K.; Iverson, Samuel A.; Cardona, Carol J.; Boyce, Walter M.; Dudley, Joseph P.

doi:10.3791/2829

Cited by 13 publications

(13 citation statements)

References 13 publications

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“…[ 16 ] Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents for the detection of avian influenza virus has brought on-site remote testing to a practical goal. [ 8 ] Dry-reagent-based PCR was developed for the rapid on-site detection of microbial pathogens such as blackleg of ruminants caused by Clostridium chauvoei . Basic PCR reagents (bovine serum albumin, PCR buffer, MgCl 2 , and primers) were dried on polyolefin matrices, showed stability at ambient temperatures for up to 10 months without any loss of functionality, eliminated PCR error, and saved time.…”

Section: Discussionmentioning

confidence: 99%

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Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction

et al. 2018

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BACKGROUND:Nosocomial infections are often caused by multidrug-resistant bacteria and the incidence is increasing. Acinetobacter, a Gram-negative bacillus, is commonly associated with the use of intravascular catheterization and airway intubation. Polymerase chain reaction (PCR) for identification of Acinetobacter baumannii from samples has been standardized that use conventional wet-reagent mix. We have designed and optimized a dry-reagent mix for identification of Acinetobacter species by PCR. The dry-reagent mix can be stored at room temperature, has less chances of contamination, and thus can be used at point-of-care diagnosis.AIM AND OBJECTIVE:The present work was focused on comparing the sensitivity and specificity of dry-reagent PCR mix over conventional wet-reagent PCR mix for identification of Acinetobacter species.MATERIALS AND METHODS:Conventional wet-reagent mix based and dry-reagent mix based PCR were carried out for the DNA isolated from Acinetobacter species. The latter was also applied directly on bacterial growth without prior DNA extraction process. Equal numbers of bacterial isolates other than Acinetobacter species were also subjected to identification by the same protocols for determining the sensitivity and specificity of the test.RESULTS:The Acinetobacter species showed amplification of the target rpoB gene and the band was observed at 397 bp. The dry-reagent PCR mix results matched completely with the conventional wet-reagent PCR mix assay. All the non-Acinetobacter isolates were negative for the PCR. This indicates that the test is highly specific. The dry-reagent mix also contained an enzyme resistant to PCR inhibitors and capable of amplifying DNA directly from cells.CONCLUSION:Performance of dry-reagent PCR mix without the need for DNA extraction and preparation of a PCR mix proved to be more sensitive and reduce the handling error, minimizes the time, manual work, and skilled labor.

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Section: Discussionmentioning

confidence: 99%

“…They state that the use of dry-reagent mix is cost-effective, time efficient, stable at room temperature, less demanding, and easy to handle, thus useful in low-resource settings. [ 7 8 9 10 11 ]…”

Section: Introductionmentioning

confidence: 99%

Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction

et al. 2018

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“…There are other platforms that do not include nucleic acid extraction step (need to be done separately), such as genesig (Primerdesign Ltd, United Kingdom), Genedrive (Epistem Ltd, Manchester, United Kingdom), Cepheid SmartCycler (Cepheid), T-COR 8 (Tetracore), and R.A.P.I.D. (Idaho Technologies) (Takekawa et al, 2010(Takekawa et al, ,2011. The genesig is now supplying lyophilized qPCR assay kits for 62 bovine, 42 equine, 47 porcine, 60 avian, 40 canine, and 26 feline different pathogens.…”

Section: Point-of-care Diagnosticsmentioning

confidence: 99%

Biotechnological innovations in farm and pet animal disease diagnosis

Malik

Verma

Kumar

et al. 2020

Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries

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“…Tomlinson et al freezedried PCR mixes for the detection of Phytophthora ramorum, which could be stored at room temperature for 20 weeks [12]. Takekawa et al freeze-dried a PCR mix for the detection of avian influenza virus in wild birds, but did not report the preservation time [13,14].…”

Section: Introductionmentioning

confidence: 99%

Room-temperature-storable PCR mixes for SARS-CoV-2 detection

Wang

Zhong

et al. 2020

Clinical Biochemistry

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A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) emerged in late 2019, causing an outbreak of pneumonia [coronavirus disease 2019 (COVID-19)] globally. Although the use of ready-made reaction mixes can enable more rapid PCR-based diagnosis of COVID-19, the need to transport and store these mixes at low temperatures presents challenges to already overburdened logistics networks. Methods: Here, we present an optimized freeze-drying procedure that allows SARS-CoV-2 PCR mixes to be transported and stored at ambient temperatures, without loss of activity. Additive-supplemented PCR mixes were freeze-dried. The residual moisture of the freeze-dried PCR mixes was measured by Karl-Fischer titration. Results: We found that the freeze-dried PCR mixes with~1.2% residual moisture are optimal for storage, transport, and reconstitution. The sensitivity, specificity, and repeatability of the freeze-dried reagents were similar to those of freshly prepared, wet reagents. The freeze-dried mixes retained activity at room temperature (18~25°C) for 28 days, and for 14 and 10 days when stored at 37°C and 56°C, respectively. Conclusion: The uptake of this approach will ease logistical challenges faced by transport networks and make more cold storage space available at diagnosis and hospital laboratories.

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Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Cited by 13 publications

References 13 publications

Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction

Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction

Biotechnological innovations in farm and pet animal disease diagnosis

Room-temperature-storable PCR mixes for SARS-CoV-2 detection

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