Comparison of various RNA extraction methods, cDNA preparation and isolation of calmodulin gene from a highly melanized isolate of apple leaf blotch fungus Marssonina coronaria

ABSTRACTExtraction of high-quality mRNA fromCryptosporidium parvumis a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure forCryptosporidiumdetection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection ofCryptosporidiumwith oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, andSalmonella entericaserovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed thatSalmonellacells most efficiently relieved binding of RNA. With the inclusion ofSalmonelladuring extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102oocysts g−1of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104C. parvumoocysts g−1soil for sandy, loamy, and clay samples, respectively.

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Rapid Virus Detection Procedure for Molecular Tracing of Shellfish Associated with Disease Outbreaks

Journal of Food Protection ◽

10.4315/0362-028x-70.4.967 ◽

2007 ◽

Vol 70 (4) ◽

pp. 967-974 ◽

Cited By ~ 25

Author(s):

ANA MARIA de RODA HUSMAN ◽

FROUKJE LODDER-VERSCHOOR ◽

HAROLD H. J. L. van den BERG ◽

FRANÇOISE S. LE GUYADER ◽

HILDE van PELT ◽

...

Keyword(s):

Extraction Method ◽

Rna Extraction ◽

Virus Detection ◽

Disease Outbreaks ◽

Extraction Methods ◽

Virus Rna ◽

Pathogenic Viruses ◽

Rna Extraction Methods ◽

Digestive Glands ◽

Outbreak Situation

Detection of pathogenic viruses in oysters implicated in gastroenteritis outbreaks is often hampered by time-consuming, specialist virus extraction methods. Five virus RNA extraction methods were evaluated with respect to performance characteristics and sensitivity on artificially contaminated oyster digestive glands. The two most promising procedures were further evaluated on bioaccumulated and naturally contaminated oysters. The most efficient method was used to trace the source in an outbreak situation. Out of five RNA extraction protocols, PEG precipitation and the RNeasy Kit performed best with norovirus genogroup III–spiked digestive glands. Analyzing 24-h bioaccumulated oysters revealed a slightly better sensitivity with PEG precipitation, but the RNeasy Kit was less prone to concentrate inhibitors. The latter procedure demonstrated the presence of human noroviruses in naturally contaminated oysters and oysters implicated in an outbreak. In this outbreak, in four out of nine individually analyzed digestive glands, norovirus was detected. In one of the oysters and in one of the fecal samples of the clinical cases, identical norovirus strains were detected. A standard and rapid virus extraction method using the RNeasy Kit appeared to be most useful in tracing shellfish as the source in gastroenteritis outbreaks, and to be able to make effective and timely risk management decisions.

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Comparison of four RNA extraction methods for the detection of small round structured viruses in faecal specimens

Journal of Virological Methods ◽

10.1016/0166-0934(95)01966-9 ◽

1996 ◽

Vol 57 (2) ◽

pp. 195-201 ◽

Cited By ~ 56

Author(s):

A.D. Hale ◽

J. Green ◽

D.W.G. Brown

Keyword(s):

Rna Extraction ◽

Extraction Methods ◽

Rna Extraction Methods

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Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-Time and Conventional Reverse Transcription–PCR

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870501700609 ◽

2005 ◽

Vol 17 (6) ◽

pp. 574-578 ◽

Cited By ~ 41

Author(s):

Ming Y. Deng ◽

He Wang ◽

Gordon B. Ward ◽

Tammy R. Beckham ◽

Thomas S. McKenna

Keyword(s):

Real Time ◽

Classical Swine Fever Virus ◽

Rna Extraction ◽

Classical Swine Fever ◽

Extraction Methods ◽

Rt Pcr ◽

Total Rna ◽

Reverse Transcription Pcr ◽

Rna Extraction Methods ◽

Positive Results

Six RNA extraction methods, i.e., RNAqueous kit, Micro-to-midi total RNA purification system, NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and TRIzol LS reagent, were evaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6 extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription–PCR (RT-PCR) for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positive results in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encoding β-actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCR assays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methods are more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood and tissues. However, some of the 6 reagents offer certain advantages not common to all 6 extraction procedures. For example, RNA extracted by the TRIzol LS reagent constantly had the highest yield; that by the RNAqueous kit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElute mammalian total RNA kit was most likely to be free of contaminations with genomic DNA.

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