scholarly journals Sensitivity of polymerase chain reaction for detection of known aliquots of Trypanosoma cruzi in the blood of mice: an in vitro study

2002 ◽  
Vol 35 (5) ◽  
pp. 487-490 ◽  
Author(s):  
Rozália F. Campos ◽  
Juracy B. Magalhães ◽  
Eliana A.G. Reis ◽  
Mitermayer G. Reis ◽  
Sonia G. Andrade
Keyword(s):  
Polymerase Chain Reaction ◽  
Trypanosoma Cruzi ◽  
In Vitro Study ◽  
High Sensitivity ◽  
Positive Control ◽  
Chain Reaction ◽  
Negative Results ◽  
Polymerase Chain ◽  
Positive Controls

To evaluate the sensitivity of polymerase chain reaction (PCR) to reveal known number of trypomastigote in the blood of mice, three separate experiments were done. First: To eight samples of 500mul of normal mice blood, one aliquot of 1, 2, 3, 4, 5, 10, and 50 trypomastigotes respectively, were added. Second and third: 10 aliquots with 1 and 10 with 2 trypomastigotes were added to samples of 500mul of normal mice blood. Positive control: 500mul of blood containing 100,000 trypomastigotes. For kDNA minicircles amplification by PCR the primers:S35 and S36 were used. PCR revealed products of 330 b.p in the positive controls. When only one sample with the aliquots of 1 or 2 trypomastigotes was examined, results were negative; results were positive with aliquots of 3 to 50 trypomastigotes. In the 2nd and 3rd experiments, 9/10 aliquots with one parasite and 9/10 with 2 trypomastigotes were positive revealing a high sensitivity of this reaction. In conclusion, the presence of one single parasite in 500mul of blood, is enough for a positive PCR. This method could be used as a complement to the various parasitological cure tests in treated mice, when low volumes of blood are individually examined.

scholarly journals Accidental infection by Trypanosoma cruzi follow-up by the polymerase chain reaction: case report

2009 ◽  
Vol 51 (5) ◽  
pp. 295-298 ◽  
Author(s):  
Andréa Tieko Kinoshita-Yanaga ◽  
Max Jean de Ornelas Toledo ◽  
Silvana Marques de Araújo ◽  
Berenice Pelizza Vier ◽  
Mônica Lúcia Gomes
Keyword(s):  
Polymerase Chain Reaction ◽  
Trypanosoma Cruzi ◽  
Blood Smear ◽  
Fresh Blood ◽  
Chain Reaction ◽  
Negative Results ◽  
Laboratory Confirmation ◽  
Polymerase Chain ◽  
Positive Results

We report a case of accidental infection by Trypanosoma cruzi in a 42-year-old female patient who presented an inoculation chagoma. Laboratory confirmation was based on examination of fresh blood, Giemsa-stained blood smear, immunoenzyme test (EIA-IgG), indirect immunofluorescence (IIF-IgM, IgG) and polymerase chain reaction (PCR). Only the PCR gave a positive result, and the EIA test was inconclusive. Two treatments with benznidazole were necessary. PCR was the only technique that continued to give positive results for approximately two months (65 days, or 2.2 months) following the second treatment and negative results from 96 days (3.2 months) to 850 days (28.3 months). We concluded that the presence of an inoculation chagoma and use of PCR were important and decisive for diagnosis and follow-up of the case.

scholarly journals Recombinant Retroviral Particles: Technology of Poduction and Application as Positive Controls for PCR Diagnostics of Dangerous Viral Infections

2020 ◽  
pp. 115-121
Author(s):  
E. G. Fomina ◽  
E. E. Grigorieva ◽  
A. S. Vladyko
Keyword(s):  
Flow Cytometry ◽  
Polymerase Chain Reaction ◽  
Viral Infections ◽  
Positive Control ◽  
Infectious Agents ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Pcr Diagnostics ◽  
Control Samples ◽  
Positive Controls

Objective. Construction of positive control samples based on recombinant retroviral particles and their application in RT-PCR diagnostic assays for RNA detection of agents of dangerous and particularly dangerous viral infections.Materials and methods. Molecular biological, genetic engineering, and immunological methods were used: polymerase chain reaction, restriction, ligation, cloning, transformation, transfection, flow cytometry.Results and discussion. Technology of positive control samples producing based on recombinant virions has been developed and tested. It includes construction of retroviral vector with cloned diagnostic sequence of the viral genome; obtaining a packaging cell line producing chimeric retroviral particles; determination of recombinant virions titer by flow cytometry and polymerase chain reaction; application of the obtained preparation as a control sample for PCR diagnostics of infectious agents. Positive controls based on retroviral vectors as carriers of genomic RNA fragments of pathogenic viruses were used in the development of PCR diagnostic kits for dangerous and particularly dangerous viral infections. Their application increased the kits quality and made it possible to exclude the work with concentrated hazardous infectious agents (Lassa virus, tick-borne encephalitis virus, lymphocytic choriomeningitis virus, Puumala virus).

scholarly journals Use of polymerase chain reaction in verification and differential diagnosis of babesiosis pathogens

2020 ◽  
Vol 11 (4) ◽  
pp. 563-567
Author(s):  
I. I. Torianyk ◽  
O. M. Tymchenko ◽  
M. O. Ostapets ◽  
N. A. Chygyrynska ◽  
S. I. Pokhyl ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Objective Assessment ◽  
Fatal Outcome ◽  
High Sensitivity ◽  
Blood Parasites ◽  
Chain Reaction ◽  
Negative Results ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Sensitivity Specificity

Today, Babesia is recognized as one of the most common blood parasites in the world, which in terms of the number of cases of invasion is second only to trypanosomes (the causative agent of African trypanosomiasis and Chagas’ disease). These microorganisms can cause parasitism in erythrocytes and hematopoietic organs. They cause an infectious process, the clinical course of which can vary from asymptomatic, subclinical, mild or moderate influenza-like forms – to severe progressive disease (fulminant form) with fatal outcome. Thus, the latter determines the significant burden of babesia for the leading branches of medicine, veterinary medicine and the economy as a whole. The presented work is devoted to the study of the prospects for verification of babesiosis causative agents by the polymerase chain reaction (PCR) method. Blood, erythrocyte suspension, homogenized tick-carriers of babesiosis, culture of Babesia spp. were used as research material (samples). In order to obtain an objective assessment, the PCR-diagnostics method was used in two formats – standard and multiplex (multi-primer). Multiple PCR testing of multiplex format using primers in model samples containing cells of different species of Babesia (B. microti, B. divergens, B. bovis, B. canis), allowed us to establish the level of reproducibility of the results of such studies, which ranged 94.6–96.4%, to determine the level of PCR sensitivity of the multiplex format for detection/identification of human pathogenic babesia (B. microti, B. divergens and B. venatorum). It is established that the advantages of the PCR-diagnostic method of babesiosis pathogens in the samples of the studied biomaterial were: speed of research (2–4 hours); high sensitivity, specificity, reproducibility of Babesia detection results, prospects of species identification, differentiation with apicomplex spores (Plasmodium falciparum, Toxoplasma). In view of the above, the PCR method is recommended for use in cases of persistent suspicion of babesiosis infection (in cases of negative results of microscopic/cytological studies, to identify asymptomatic, subclinical and chronic forms of babesiosis, verification of active invasion in seropositive individuals and for Babesia species and their differentiation).

scholarly journals Amplification of human beta-actin gene by the reverse transcriptase-polymerase chain reaction: implications for assessment of RNA from formalin-fixed, paraffin-embedded material.

1996 ◽  
Vol 44 (10) ◽  
pp. 1205-1207 ◽  
Author(s):  
A Dakhama ◽  
V Macek ◽  
J C Hogg ◽  
R G Hegele
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Housekeeping Gene ◽  
Chain Reaction ◽  
Viral Genomes ◽  
Negative Results ◽  
Beta Actin ◽  
Target Nucleic Acid ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain

The polymerase chain reaction (PCR) is a powerful method that allows enzymatic amplification of rate target nucleic acid sequences. It has been applied to the amplification of viral genomes from paraffin-embedded pathology specimens. However, interpretation of negative results requires amplification of a housekeeping gene such as beta-actin. In the present study we used specific oligonucleotide primers previously designed to amplify both the genomic DNA and the mRNA transcript from paraffin-embedded tissue. These products have predicted sizes of 250 BP and 154 BP, respectively, but our results showed that PCR amplification only (without reverse transcription) unexpectedly generated the 154-BP product. Further investigation of the nature of this product demonstrated that it originated from the amplification of DNA, not RNA. We conclude that the 154-BP product generated by these primers cannot be exclusively considered as beta-actin RNA product and should not be used to assess successful extraction of RNA, to ascertain its integrity, or to normalize for the total amount of RNA assayed by RT-PCR from paraffin-embedded tissue.

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