Assessment of different commercial RNA-extraction and RT-PCR kits for detection of hepatitis A virus in mussel tissues.
In the present study, the efficiency of several nucleic acid extraction and RT-PCR commercial kits for the detection of hepatitis A virus (HAV) from seeded mussel tissue samples was evaluated in comparison with the "in-house" method used currently in our laboratory. The best results were achieved with Total Quick RNA Cells & Tissues version mini (Talent) for RNA extraction and the Superscript One-Step RT-PCR System (Life Technologies) for the RT-PCR reaction, obtaining a detection limit of 0.1-1pfu/mg of mussel tissue. A slightly lower sensitivity (in 1logunit) was achieved using the Rneasy plant mini kit (Qiagen) and the Total Quick RNA Cells & Tissues version maxi in combination with the Superscript RT-PCR system. The conventional method usually employed in our laboratory resulted in a sensitivity of 300pfu/mg of tissue. Taken together, these findings indicate that the combination of Total Quick RNA Cells & Tissues version mini and Superscript One-Step RT-PCR System cannot only improve significantly the sensitivity for the HAV detection from mussel, but are also labor and time saving and easy to standardize.
Ribao
Cristina
C
Departamento de Microbiologi;a y Parasitologi;a, Facultad de Biologi;a, Universidad de Santiago de Compostela, Santiago de Compostela 15782, Spain.
Torrado
Ignacio
I
Vilariño
M Luz
ML
Romalde
Jesús L
JL
D000818:Animals; D001483:Base Sequence; D049872:Bivalvia / Q000821:virology*; D005516:Food Microbiology; D017957:Hepatitis A Virus, Human / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000821:virology*; D014773:Virology / Q000379:methods* / Q000706:statistics & numerical data
D012367:RNA, Viral