Evaluation of different RNA-extraction kits for sensitive detection of hepatitis A virus in strawberry samples.
The efficiency of different commercial RNA extraction kits for the detection of hepatitis A virus (HAV) from seeded strawberry samples was assessed by standard RT-PCR and real time RT-PCR (RT-qPCR). The best results with standard RT-PCR were achieved with Aurum™ Total RNA extraction kit (BioRad), obtaining a detection limit of 5-6.25 pfu/mg of tissue. A slightly lower sensitivity was rendered by the RNeasy® Plant mini kit (Qiagen) (10-12.5 pfu/mg tissue), while the Total Quick RNA Cells and Tissues kit version mini (Talent) rendered a detection limit of 50-100 pfu/mg of tissue. The other tested commercial kits showed worse detection limits (>500 pfu/mg). With RT-qPCR and ten fold diluted RNA all the kits showed an increase of sensitivity, detecting the kits from Qiagen, Talent and BioRad down to 0.05 pfu/mg of strawberry homogenate. These findings indicate that the use of Aurum™ Total RNA extraction kit, with standard RT-PCR technique or RT-qPCR, can not only be labor and time saving but also helpful to improve the sensitivity for the HAV detection from fruits and to facilitate the standardization of detection methods among laboratories.
Istituto di Virología, Facoltà di Medicina e Chirurgia, Università degli Studi di Milano, Milano, Italy.
Silvia
S
Bianchi
Anna
A
Dal Vecchio
María Luz
ML
Vilariño
Jesús L
JL
Romalde
D005504:Food Analysis; D005516:Food Microbiology; D031985:Fragaria / Q000821:virology*; D030041:Hepatitis A virus / Q000235:genetics / Q000302:isolation & purification*; D012313:RNA / Q000378:metabolism; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D012313:RNA