PCR Test System Research Articles

Improvement of Oligonucleotide Primers Synthesis Technology for PCR Test Systems Production

The investigations carried out resulted in the improvement of technology of synthesis of the oligonucleotide primers, components of the PCR test systems produced in the RARI Microbe. The final product output was demonstrated to increase by 93 % when 25 % aquatic ammonium solution was used for ammonolysis, and by 16 % if solutions Cap A N1 and Cap B N 3 were used at the stage of capping. Application of the improved technology of oligonucleotide synthesis for gene diagnostic preparations production will permit to increase the amount of the output products and reduce expenses for high-priced reagents.

Rapid quantitation of cytomegalovirus DNA in whole blood by a new molecular assay based on automated sample preparation and real-time PCR

Quantitation of cytomegalovirus (CMV) DNA in whole blood samples has gained significance in the routine diagnostic laboratory. In this study, the analytical performance of the artus™ CMV RG PCR kit in conjunction with automated sample preparation on the new QIAsymphony™ SP instrument was evaluated. Clinically referred samples were tested and results compared to those obtained with the routinely used molecular test system. Accuracy testing showed results to be within ±0.2 log(10) unit of the expected panel results. The assay was linear (R=0.9972) from a lower quantification limit of 148 copies/ml to 1.3×10(7) copies/ml. The between-day imprecision CV was 8-63%, and the within-run imprecision CV was 16-61%. When 100 clinically referred samples were tested, results obtained with the new test system showed an acceptable concordance with those obtained with the routinely used easyMAG™ sample preparation and CMV HHV6,7,8 R-gene™ test system. Discrepant results were found with low-titer samples containing CMV DNA concentrations under the lower limit of quantification or within half a log unit above. The time to results was comparable for both test systems. The QIAsymphony™ sample preparation and artus™ CMV RG PCR test system allows for a rapid, sensitive, precise, and accurate high-throughput quantitation of CMV DNA in whole blood in the routine diagnostic laboratory.

Development of a test system for simultaneous detection of HIV RNA and Hepatitis C virus (HCV) and DNA of Hepatitis B virus (HBV) in blood samples

Background The aim of this work was to create a PCR test systems for simultaneous detection of HIV RNA, HCV RNA and DNA of HBV, which will have high sensitivity and high bandwidth. The issue of viral safety of blood products is of particular relevance in relation to the epidemiological situation with regard to viral hepatitis and HIV infection. The use of serological tests for screening of blood products has significantly reduced the risk of transmission of these infections in the transfusion. But from the moment of infection until the appearance of antibodies to the virus may be a considerable period, known as «serological window». In this situation, great care may have tests to detect the virus itself or its components. Using molecular genetics methods of diagnosis helped reduce the period of «serological window» and improve the safety of transfusion. The actual development of low-cost highly sensitive tests, allowing both to detect HIV and hepatitis viruses B and C, increase the capacity of blood transfusion stations in the molecular genetic studies. This involves the basic requirements for test systems for screening of donated blood the highest analytical sensitivity.

Detection and quantitation of Epstein-Barr virus (EBV) DNA in EDTA whole blood samples using automated sample preparation and real time PCR

Detection and quantitation of Epstein-Barr virus (EBV) DNA in EDTA whole blood samples has gained significance in the routine diagnostic laboratory. In this study, the analytical and clinical performance of the artus EBV RG PCR kit in conjunction with automated sample preparation on the QIAsymphony SP instrument was evaluated. When the accuracy of the new test system was tested, all results were found to be within +/-0.5 log(10) unit of the expected panel results. Determination of linearity showed a quasilinear curve over 4 log units. The lower limit of detection was determined to be 391 EBV DNA copies/mL in EDTA whole blood. The between day imprecision ranged from 18% to 66%, and the within run imprecision ranged from 11% to 50%. When clinical samples were tested and the results compared with those obtained with the routinely used easyMAG sample preparation and EBV R-gene test system, 60 samples tested positive and 31 samples tested negative by both assays. Nineteen samples were found to be positive using the QIAsymphony sample preparation and artus EBV RG PCR test system only, and no samples tested positive with the routinely used test system only. The QIAsymphony sample preparation and artus EBV RG PCR test system is suitable for the detection and quantitation of EBV DNA in EDTA whole blood in the routine diagnostic laboratory.

Torque Teno Virus (TTV) distribution in healthy Russian population

BackgroundTorque teno virus (TTV) is a circular, single-stranded DNA virus that chronically infects healthy individuals of all ages worldwide. There is a lot of data on the prevalence and genetic heterogeneity of TTV in healthy populations and in patients with various diseases now available. However, little is known about TTV load among healthy human population. In this study we analyzed TTV load in the group of 512 Russian elite athletes, who are supposed to be, by some standards, the healthiest part of the human population.ResultsThe prevalence rate of TTV among the Russian Olympic Reserve members was 94% (for test sensitivity about 1000 genome equivalents per 1 ml of blood). Quantities varied from 103 (which corresponded to detection limit) to 1010 copies per 1 ml of blood, with median at 2.7 × 106 copies.ConclusionAbout 94% of healthy individuals in Russian population have more than 1000 TTV genome copies per 1 ml of blood. This result exceeds the previously published data, and can be explained by either more sensitive PCR test system or by higher TTV distribution in Russian population or both. TTV viral load neither depends on gender, nor age.

Real-time PCR in clinical microbiology: verification, validation, and contamination control

Real-time PCR is increasingly utilized in the clinical microbiology laboratory setting. Availability of a single set of guidelines for real-time PCR testing which incorporate all the necessary verification and validation recommendations set forth by accreditation agencies would be beneficial to laboratories acquiring this new technology. Presently, the challenges to the clinical microbiology laboratory for introducing real-time PCR test systems include what verification experiments are required and how many and which types of specimens should be tested. In addition, based on the regulations, the laboratory must determine what constitutes an acceptable quality control program. Contamination of specimens or samples in real-time PCR testing also needs to be addressed. Although real-time PCR test systems are closed systems, contamination from specimen-to-specimen or from previously-amplified product can lead to false-positive results causing delays in clinical diagnosis, along with wasting valuable time and resources.

A 4-nucleotide deletion in intron 2 of the HPA 1a allele of GPIIIa interferes with the PCR-SSP genotyping

Typing of platelet specific antigens by polymerase chain reaction using allele-specific primers (PCR-SSP) is a valuable tool in diagnosing alloimmune thrombocytopenia. To avoid post-PCR processing of the samples, we have adopted the Taqman™ allelic discrimination technique for typing in the HPA 1 system. By comparing the results of this test system with PCR-SSP, we found a discrepancy in one sample out of 1000. This sample was HPA 1bb in PCR-SSP and HPA 1ab in the Taqman™ assay. The HPA 1ab phenotype was confirmed with two assays: first, monoclonal immobilization of platelets antigens assay (MAIPA) using antibody with HPA 1a and HPA 1b specificity and, second, flow cytometric analysis of the platelets with the same antisera. In addition, DNA sequencing of the polymorphic site related to the HPA 1 system revealed a heterozygous pattern. However, when the region in intron 2 of the GPIIIa gene corresponding to the consensus primer in the PCR-SSP assay was sequenced, we found a 4-nucleotide deletion in the HPA 1a allele interfering with the 3′ end of the consensus primer. Consequently, the amplification of this allele could not take place. Although this gene defect is located in an intron area, and thus do not have any significance for protein expression, it is located so close to the HPA 1 polymorphic site that it may influence on test systems designed to type for HPA 1 (Skogen et al. Transfusion 1994; 34: 955–960). Therefore, we strongly suggest avoiding this region of the gene when choosing primers/probes in the construction of PCR test systems for HPA 1.

SOME SCIENTIFIC RESULTS FOR THE 2018 YEAR ALL-RUSSIAN RESEARCH INSTITUTES UNDER SCIENTIFIC AND METHODOLOGICAL GUIDANCE BY SI-BERIAN BRANCH OF RUSSIAN ACADEMY OF SCIENCE

The article provides information of the research in the following areas: economics and land relations, agriculture, plant growing and breeding, feed production, livestock technology, veterinary medicine, mechanization, automation and electrification, processing and storage of agricultural products. As a result of the state assignment, Siberian scientists have developed: “A strategy for the socio-economic development of the agro-industrial complex of the Siberian federal district in the context of globalization and integration”, “Methodology and methods for assessing the ecological-resource potential of agricultural landscapes”, methods for the effective use of nitrogen fertilizers in the cultivation of spring wheat in traditional minimum and no-till technologies, “Guidelines for the restoration of highly degraded, deserted arable land of the arid zone of Central Siberia”, “Technology creating hayfields from annual leguminous and grain grass mixtures ”. More than 10 new crop varieties have been entered into the State Register of Protected Breeding Achievements. Patents for breeding achievements have been received: Siberian livestock (Sibiriachka) and Altai-Ussuri breed of sika deer (Cervus nippon). In the field of veterinary medicine, a diagnostic PCR test system was developed for detecting the DNA of microorganisms Listeria monocytogenes, a computer program "Assessing the relationship of solar activity with animal morbidity indicators", "A system of measures for the prevention and control of brucellosis in farm animals in the Republic of Tyva." In the field of storage and processing of agricultural products, a recipe for raw cakes using vegetable raw materials and a recipe for sauce of rowan (Sorbus aucuparia) have been proposed.