scholarly journals Optimization of DNA Extraction from Field-Collected Mammalian Whole Blood on Filter Paper for Trypanosoma cruzi (Chagas Disease) Detection

Pathogens ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1040
Author(s):  
Bonnie E. Gulas-Wroblewski ◽  
Rebecca B. Kairis ◽  
Rodion Gorchakov ◽  
Anna Wheless ◽  
Kristy O. Murray
Keyword(s):  
Trypanosoma Cruzi ◽  
Filter Paper ◽  
Whole Blood ◽  
Neglected Tropical Diseases ◽  
Cost Effective ◽  
Proteinase K ◽  
Type I ◽  
Filter Strips ◽  
Materials Used ◽  
Commercial Kits

Blood filter paper strips are cost-effective materials used to store body fluid specimens under challenging field conditions, extending the reach of zoonotic pathogen surveillance and research. We describe an optimized procedure for the extraction of parasite DNA from whole blood (WB) stored on Type I Advantec Nobuto strips from both experimentally spiked and field-collected specimens from canine and skunks, respectively. When comparing two commercial kits for extraction, Qiagen’s DNeasy Blood & Tissue Kit performed best for the detection of parasite DNA by PCR from Trypanosoma cruzi-spiked canine WB samples on Nobuto strips. To further optimize recovery of β-actin from field-collected skunk WB archived on Nobuto strips, we modified the extraction procedures for the Qiagen kit with a 90 °C incubation step and extended incubation post-addition of proteinase K, a method subsequently employed to identify a T. cruzi infection in one of the skunks. Using this optimized extraction method can efficaciously increase the accuracy and precision of future molecular epidemiologic investigations targeting neglected tropical diseases in field-collected WB specimens on filter strips.

scholarly journals Detection of Anti-Human T-Lymphotropic Virus Type I Antibody in Whole Blood by a Novel Counting Immunoassay

2003 ◽  
Vol 49 (2) ◽  
pp. 275-280 ◽  
Author(s):  
Kazunari Yamaguchi ◽  
Yuji Yonemura ◽  
Hiroaki Okabe ◽  
Yoichi Takahama ◽  
Shinya Nagai ◽  
...  
Keyword(s):  
Virus Type ◽  
Whole Blood ◽  
Blood Cells ◽  
Measuring Method ◽  
Sample Volume ◽  
Latex Agglutination ◽  
Blood Collection ◽  
Type I ◽  
Latex Particles ◽  
T Lymphotropic Virus

Abstract Background: Assays to screen for and confirm the presence of the antibody for human T-lymphotropic virus type I (HTLV-I) are currently performed with serum or plasma. We developed and evaluated a new counting immunoassay (CIA) for the detection of HTLV-I antibody in whole blood, using recombinant and synthetic peptide antigens. Methods: We assessed the CIA for detection of HTLV-I antibody in whole blood and plasma. The CIA is an immunity-measuring method that combines latex agglutination with particle-counting technology. The numbers of agglutinated latex particles, single latex particles, and blood cells in a sample are measured based on differences in particle size between latex particles and blood cells. Results: The CIA and ELISA methods were in agreement for all 24 plasma samples tested, including those from 6 patients with HTLV-I-associated diseases, 6 HTLV-I carriers, and 12 HTLV-I antibody-negative individuals. The concordance between the ELISA (plasma) and the CIA (whole blood) for samples from 24 patients was 100%. The concordance between a particle agglutination method (plasma) and the CIA (plasma or whole blood) for 1065 patients was 99.5%. The concordance between results obtained for 1065 pairs of plasma and whole blood samples with the CIA method was 100%. HTLV-I antibody in whole blood was stable for 3 days after blood collection. With this CIA method, results were available within 15 min. Conclusions: The CIA method can be used in screening for HTLV-I. The use of whole blood rather than serum or plasma reduces the sample volume and number of blood collections required, as well as assay time.

scholarly journals Nucleotide Polymorphisms in the α2 Gene Define Multiple Alleles That Are Associated With Differences in Platelet α2β1 Density

Blood ◽  
1998 ◽  
Vol 92 (7) ◽  
pp. 2382-2388 ◽  
Author(s):  
Marcie Kritzik ◽  
Brian Savage ◽  
Diane J. Nugent ◽  
Sentot Santoso ◽  
Zaverio M. Ruggeri ◽  
...  
Keyword(s):  
Whole Blood ◽  
Type I Collagen ◽  
Type I ◽  
Nucleotide Polymorphisms ◽  
Coding Region ◽  
Platelet Surface ◽  
Multiple Alleles ◽  
Increased Risk ◽  
Allelic Differences

Three allelic differences in the α2 gene are associated with expression levels of the α2β1 integrin on the platelet surface. We have previously defined two linked silent polymorphisms in the α2 gene coding region at nucleotides 807 (C or T) and 873 (G or A). We have now identified one rarer nucleotide polymorphism in the coding region at nucleotide 837 (T or C) and four additional linked polymorphisms within the introns that flank these coding sequences. Moreover, we have determined that the alloantigenic Br polymorphism, which resides in a distal coding region at nucleotide 1648, is also linked to the 837 polymorphism. Thus, three α2 gene alleles, defined by eight nucleotide polymorphisms, have now been discovered. Allele 1 (807T/837T/873A/Brb) is associated with increased levels of α2β1; allele 2 (807C/837T/873G/Brb) and allele 3 (807C/837C/873G/Bra) are each associated with lower levels of α2β1. Finally, we also show here that the rate of platelet attachment to type I collagen in whole blood under conditions of high shear rate (1,500/s) is proportional to the density of α2β1 receptors on the platelet surface. Thus, the density of platelet α2β1 could have an important impact on platelet adhesion to collagen in whole blood and therefore on platelet function in vivo, contributing to an increased risk of thrombosis or to bleeding in relevant disease states.

scholarly journals Detection of Pneumocystis carinii DNA in Blood Specimens from Human Immunodeficiency Virus-Infected Patients by Nested PCR

1999 ◽  
Vol 37 (1) ◽  
pp. 127-131 ◽  
Author(s):  
Meja Rabodonirina ◽  
Laurent Cotte ◽  
André Boibieux ◽  
Karine Kaiser ◽  
Martine Mayençon ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Whole Blood ◽  
Nested Pcr ◽  
Large Subunit ◽  
Pneumocystis Carinii ◽  
Proteinase K ◽  
Rrna Gene ◽  
Immunodeficiency Virus ◽  
Blood Specimens ◽  
Large Subunit Rrna

The detection of Pneumocystis carinii DNA in blood by PCR could be useful for studying the natural history of pneumocystosis and could also be a noninvasive diagnostic method. The results of previous studies are nevertheless conflicting. In our study, we compared three commercially available DNA extraction kits (GeneReleaser, QIAamp Tissue Kit, and ReadyAmp Genomic DNA Purification System) and proteinase K and proteinase K-phenol-chloroform treatments for the extraction of P. carinii DNA from dilutions of a P. carinii f. sp.hominis cyst suspension mixed with human whole blood. A rapid and simple nested PCR protocol which amplifies a portion of the mitochondrial large-subunit rRNA gene was applied to all the extraction products. The QIAmp Tissue Kit was the most effective kit for the isolation of amplification-ready P. carinii DNA and was used with nested PCR for the testing of whole-blood specimens from 35 immunocompetent control patients and 84 human immunodeficiency virus (HIV)-infected patients investigated for pulmonary disease and/or fever. In HIV-infected patients, P. carinii DNA was detected by nested PCR in blood samples from 3 of 14 patients with microscopically proven P. carinii pneumonia, 7 of 22 patients who were considered to be colonized with P. carinii, and 9 of 48 patients who were neither infected nor colonized with P. carinii. P. carinii DNA was not detected in blood specimens from the 35 immunocompetent patients. P. carinii DNA in blood might represent viable P. carinii organisms or DNA complexes released from pulmonary phagocytes. In conclusion, P. carinii DNA may be detected in whole blood from HIV-infected patients, but the nature and the meaning of the circulating form of P. carinii remain to be established.

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