A viability assay combining palladium compound treatment with quantitative PCR to detect viable Mycobacterium avium subsp. paratuberculosis cells

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F67985904%3A_____%2F22%3A00556873" target="_blank" >RIV/67985904:_____/22:00556873 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00027162:_____/22:N0000037 RIV/00216224:14310/22:00125562

Výsledek na webu

<a href="https://www.nature.com/articles/s41598-022-08634-x" target="_blank" >https://www.nature.com/articles/s41598-022-08634-x</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1038/s41598-022-08634-x" target="_blank" >10.1038/s41598-022-08634-x</a>

Jazyk výsledku

angličtina

Název v původním jazyce

A viability assay combining palladium compound treatment with quantitative PCR to detect viable Mycobacterium avium subsp. paratuberculosis cells

Popis výsledku v původním jazyce

Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogenic bacterium causing the paratuberculosis, chronic and infectious disease common particularly in wild and domestic ruminants. Currently, culture techniques to detect viable MAP are still used most commonly, although these require a long incubation period. Consequently, a faster molecular method for assessing MAP cell viability based on cell membrane integrity was introduced consisting of sample treatment with the intercalation dye propidium monoazide (PMA) followed by quantitative PCR (qPCR). However, the PMA-qPCR assay is complicated by demanding procedures involving work in a darkroom and on ice. In this study, we therefore optimized a viability assay combining sample treatment with palladium (Pd) compounds as an alternative viability marker to PMA, which does not require such laborious procedures, with subsequent qPCR. The optimized Pd-qPCR conditions consisting of 90 min exposure to 30 mu M bis(benzonitrile)dichloropalladium(II) or 30 mu M palladium(II)acetate at 5 degrees C and using ultrapure water as a resuspension medium resulted in differences in quantification cycle (Cq) values between treated live and dead MAP cells of 8.5 and 7.9, respectively, corresponding to approximately 2.5 log units. In addition, Pd-qPCR proved to be superior to PMA-qPCR in distinguishing between live and dead MAP cells. The Pd-qPCR viability assay thus has the potential to replace time-consuming culture methods and demanding PMA-qPCR in the detection and quantification of viable MAP cells with possible application in food, feed, clinical and environmental samples.

Název v anglickém jazyce

A viability assay combining palladium compound treatment with quantitative PCR to detect viable Mycobacterium avium subsp. paratuberculosis cells

Popis výsledku anglicky

Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogenic bacterium causing the paratuberculosis, chronic and infectious disease common particularly in wild and domestic ruminants. Currently, culture techniques to detect viable MAP are still used most commonly, although these require a long incubation period. Consequently, a faster molecular method for assessing MAP cell viability based on cell membrane integrity was introduced consisting of sample treatment with the intercalation dye propidium monoazide (PMA) followed by quantitative PCR (qPCR). However, the PMA-qPCR assay is complicated by demanding procedures involving work in a darkroom and on ice. In this study, we therefore optimized a viability assay combining sample treatment with palladium (Pd) compounds as an alternative viability marker to PMA, which does not require such laborious procedures, with subsequent qPCR. The optimized Pd-qPCR conditions consisting of 90 min exposure to 30 mu M bis(benzonitrile)dichloropalladium(II) or 30 mu M palladium(II)acetate at 5 degrees C and using ultrapure water as a resuspension medium resulted in differences in quantification cycle (Cq) values between treated live and dead MAP cells of 8.5 and 7.9, respectively, corresponding to approximately 2.5 log units. In addition, Pd-qPCR proved to be superior to PMA-qPCR in distinguishing between live and dead MAP cells. The Pd-qPCR viability assay thus has the potential to replace time-consuming culture methods and demanding PMA-qPCR in the detection and quantification of viable MAP cells with possible application in food, feed, clinical and environmental samples.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/QK1810212" target="_blank" >QK1810212: Rychlé, komplexní a multiplexní metody pro simultánní detekci původců alimentárních onemocnění v potravinách živočišného a rostlinného původu</a><br>

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2022

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Scientific Reports

ISSN

2045-2322

e-ISSN

2045-2322

Svazek periodika

12

Číslo periodika v rámci svazku

1

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

9

Strana od-do

4769

Kód UT WoS článku

000780307600006

EID výsledku v databázi Scopus

2-s2.0-85126728981