Lysis buffer (10 mM tris-HCl, 50 mM KCl, 2.5 mM MgCl2, 0.45% NP40, 0.45% Tween 20, 0.1 mg/ml gelatin)
Lysis of synovial tissue specimens for DNA extraction.
3
Outer primers P1 and P6
Amplification of a fragment of the DNA sequence coding for the nonstructural protein (NS1) of parvovirus B19 by nested PCR.
4
Inner nested primers P2 and P5
Second amplification in nested PCR to target the nonstructural protein (NS1) of parvovirus B19.
5
Ethidium bromide
Staining of the 103 base pair (bp) diagnostic fragment for detection after agarose gel electrophoresis.
6
Primers PC04 and GH20
Amplification of a 268-bp fragment of the beta-globin gene to test the absence of inhibitors of Taq DNA polymerase and estimate the quantity of DNA extracted from synovial membrane specimens.
7
Anti-B19 IgM and IgG enzyme immunoassays (Biotrin, Dublin, Ireland)
Testing for the presence of anti-B19 IgM and IgG antibodies in the serum.
8
Immunoblot test (recomBlot Parvovirus B19 IgG/IgM, Mikrogen GmbH, München, Germany)
Detection of IgG antibodies against human parvovirus B19 capsid proteins and NS1 protein.
9
Outer forward primer (positions 195 to 212) and inner forward primer (positions 221 to 237)
Amplification of the B19 promoter region from clinical specimens in nested PCR.
10
Inner reverse primer (positions 494 to 475) and outer reverse primer (positions 532 to 509)
Amplification of the B19 promoter region from clinical specimens in nested PCR.
11
QIAquick PCR Purification Kit (Qiagen)
Purification of all PCR fragments.
12
pGEM-T cloning Kit (Promega)
Cloning of PCR-amplified P6 regions for sequencing.
