Comparison of four extraction methods to detect hepatitis A virus RNA in serum and stool samples

Paula, Vanessa Salete de; Villar, Lívia Melo; Gaspar, Ana

doi:10.1590/s1413-86702003000200007

Cited by 15 publications

(16 citation statements)

References 26 publications

(29 reference statements)

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“…Early protocols for RNA extraction from serum or stool included proteinase K digestion followed by phenolchloroform extraction and ethanol precipitation (167,199). Subsequently, products which used guanidinium thiocyanatephenol-chloroform (41) became commercially available for extraction of RNA and total nucleic acids (40) and increased the sensitivity and specificity of HAV detection (65,112,170). Antigen capture RT-PCR (116) and magnetic beads coated with anti-HAV (124) have been used to separate virus from potential inhibitors of reverse transcription and PCRs that are often found in environmental and stool samples.…”

Section: Molecular Detection Methodsmentioning

confidence: 99%

Diagnosis of Hepatitis A Virus Infection: a Molecular Approach

et al. 2006

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SUMMARY Current serologic tests provide the foundation for diagnosis of hepatitis A and hepatitis A virus (HAV) infection. Recent advances in methods to identify and characterize nucleic acid markers of viral infections have provided the foundation for the field of molecular epidemiology and increased our knowledge of the molecular biology and epidemiology of HAV. Although HAV is primarily shed in feces, there is a strong viremic phase during infection which has allowed easy access to virus isolates and the use of molecular markers to determine their genetic relatedness. Molecular epidemiologic studies have provided new information on the types and extent of HAV infection and transmission in the United States. In addition, these new diagnostic methods have provided tools for the rapid detection of food-borne HAV transmission and identification of the potential source of the food contamination.

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Section: Molecular Detection Methodsmentioning

confidence: 99%

Diagnosis of Hepatitis A Virus Infection: a Molecular Approach

et al. 2006

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“…A 247‐bp fragment encompassing the VP1‐2A junction of the HAV genome was amplified by nested reverse‐transcription polymerase chain reaction (RT‐PCR) using primers and conditions described previously [de Paula et al, 2003b]. RT‐PCR was performed with 10 µl extracted RNA using Superscript II H − reverse transcriptase (Invitrogen, San Diego, CA) and random hexamer primers according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Exposure to multiple subgenotypes of hepatitis a virus during an outbreak using matched serum and saliva specimens

Amado

Villar

Paula

et al. 2011

Journal of Medical Virology

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Matched serum and saliva samples were collected simultaneously from 124 subjects exposed during a hepatitis A virus (HAV) outbreak at a daycare center in Rio de Janeiro, Brazil. All samples were tested for IgM and total anti-HAV antibodies by enzyme immunoassay (EIA). HAV was detected by nested PCR in serum, saliva, and water samples employing primers for the VP1/2A region of the viral RNA; all positive products were then sequenced. The viral load of the matched samples was determined by real-time PCR using the TaqMan system. HAV-RNA was identified by nested PCR in 37.7% of the saliva samples, 29% of the serum samples, and one drinking water sample. The mean HAV viral load was similar in the serum and saliva specimens (10(3) copies/ml). HAV genotypes IA and IB were detected in both specimen types, and the water sample isolate was classified as genotype IB, indicating the existence of more than one source of infection at the daycare center. In six infected patients, a different HAV subgenotype was found in their serum than in their saliva, and this unusual pattern of mixed HAV infection was investigated further by molecular cloning followed by nucleotide sequencing. All clones derived from the saliva samples belonged to subgenotype IB and shared 96.5-100% identity. However, clones derived from their corresponding serum sample belonged to subgenotype IA and shared 90.5-100% identity. This study showed the important role that non-invasive saliva samples can play in the molecular epidemiological analysis of a hepatitis A outbreak.

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“…Since both methods employing silica presented similar results, the silica/guanidine isothiocyanate method was chosen to avoid additional steps in the DNA extraction procedure. In addition, this method is known to efficiently remove PCR inhibitors from clinical samples and it provides greater stability to the extracted nucleic acid (Romero, 1999;Rassol et al, 2002;Paula et al, 2003). Although mice are susceptible to infection with C. abortus strain S26/3, no infected mice died or presented any clinical sign during this experiment.…”

Section: Resultsmentioning

confidence: 97%