Development of a PCR assay for the detection of human herpes virus type 7.

A PCR assay has been developed to identify the DNA of the human herpes virus type 7. The search and selection of conserved regions was carried out by comparing the whole genome nucleotide sequences of HHV-7. A fragment duplicated in the HHV-7 genomes was chosen as a target for amplification. The performance of the assay was tested on a synthetic matrix and clinical samples. The developed assay has high sensitivity and specificity and showed good efficiency in detecting HHV-7 DNA in clinical samples.

Development of a reagent kit for the qualitative detection of SARS-CoV-2 virus RNA in naso- and oropharyngeal swabs by real-time RT-PCR.

The coronavirus infection continues to spread around the world. In this regard, the purpose of this work was: to develop a set of reagents for the qualitative detection of SARS-CoV-2 virus RNA. The set was developed by CJSC «Ecolab¼, 20 positive samples were used to develop the kit. The research method consisted of several stages: isolation of SARS-CoV-2 RNA, RNA reverse transcription reaction and PCR amplification of cDNA with simultaneous detection of the result in real time. The main characteristics of the kit: analytical sensitivity - 100%, specificity - 100%, accuracy - 100%. Thus, our method for diagnosing a new coronavirus infection based on real-time RT-PCR makes it possible to qualitatively and quickly detect betacoronavirus RNA in clinical material from patients and healthy individuals with suspected coronavirus infection and other symptoms of SARS.

Analysis of the domestic market for COVID-19 diagnostic kits by real-time reverse-transcription polymerase chain reaction.

COVID-19 is a disease caused by the new coronavirus SARS-CoV-2. Outbreaks were first reported in China on December 31, 2019. Exactly one month later, the WHO declared the outbreak a public health emergency of international concern, and on March 11, it was declared a pandemic. In February, the infection began to spread rapidly to various countries, with Europe declared the center. By April 17, 2020, cases had been confirmed in all subjects of the Russian Federation. At the beginning of September 2020, the number of cases exceeded one million; at November 19, two million; at December 26, three million. At February 10, 2021, four million; at May 23, five million; at July 20, six million; at September 5, seven million; at October 18, eight million; at November 13, nine million; and at December 12, 2021, ten million. The rapid spread of the virus, accompanied by a significant increase in the number of infections and deaths. A total of about 18.6 million cases were recorded at the end of the first half of 2022. The total number of deaths from coronavirus in Russia at that time was 382,313 (2.06% of all cases). The number of tests performed by various analytical methods amounted to over 274, 5 million, i.e. 1.9 million per 1 million population. The rapid spread and the increase in new infections caused by SARS-CoV-2 made it necessary to use new epidemiological and diagnostic approaches based on fast, accurate and reliable technology for detecting the infectious agent. One such virus detection method is polymerase chain reaction with reverse transcription and real-time detection of the results. The review presents the domestic market offerings of PCR diagnostic kits and provides their comparative consumer characteristics.

Detection of Y-chromosome marker in plasma of pregnant women using real time PCR: diagnostic accuracy depending on gestation age.

The evaluation of the clinical significance of the test for the detection of the Y-chromosome marker in the plasma of a pregnant woman at different stages of pregnancy by real-time PCR was carried out. The blood samples of 4616 women at 4 to 32 gestation weeks were studied. Identification of the Y-chromosome marker was carried out based on the amplification of a region of the TSPY gene. The Y-chromosome marker was unambiguously identified in 2131 samples, which accounted for 46.2% of the total number of analyzed samples. In 233 samples (5%), the Y-chromosome marker was detected with reduced reliability, and in 15 samples (0.3%), an unambiguous conclusion about the presence or absence of Y-specific DNA in plasma could not be made during the initial study. The diagnostic accuracy of the Y-chromosome marker determination in the plasma of a pregnant woman at the 4-6th gestation week was 95.5%, and from the 7th week and at later stages of pregnancy it reached 97.3-98.2%. Testing from the 7th gestation week may be recommended for reliable prenatal sex determination of the fetus by real-time PCR analysis of extracellular circulating fetal DNA.

Features of the humoral response to immunization "Gam-COVID-Vac" and in patients with COVID-19.

The paper present the results of a survey of people who have undergone immunization with a combined vector vaccine for the prevention of coronavirus infection COVID-19 «Sputnik V - Gam-COVID-Vac¼, as well as COVID-19 recovalents. Using a quantitative enzyme-linked immunosorbent assay, the levels of specific IgG were determined in persons who had had different degrees of severity before vaccination, in persons who were immuno-negative before immunization, as well as in convalescents who had undergone coronavirus infection of varying severity. The immunological targeting of antibodies against various SARS-CoV-2 proteins is considered.

About quantitative determination of the D-dimer in the blood by immunochromatographic method.

The paper presents the results of the development of a technology for the quantitative determination of D-dimer in blood with an immunochromatographic (LFIA) kit of reagents «LFIA-D-dimer¼ and instrument accounting of the results. Registration and processing of the digitized indicator of the intensity of staining of the LFIA-test strip using the LFIA-analyzer software allows quantifying the D-dimer content in the sample (in ng DDU/ml). The effectiveness of the proposed approach was evaluated on 258 clinical samples examined in the LFIA with visual and instrument accounting of the results, in comparison with the indicators of D-dimer determination in ELISA. The high reproducibility of the digitized LFIA results was shown - the coefficient of variation (CV) for samples in the range of 100-300 ng DDU/ml (near-threshold in relation to pathological values) was 2.5-5.1%; a tendency to increase CV with a further increase in the concentration of D-dimer was traced. A high correlation of the digitized LFIA result with the research data in the ELISA has been established, which makes it possible to recommend the technology for widespread use in urgent medicine.

Assessment of the state of intestinal microbiocenosis based on bacterial endotoxin and plasmalogen in elderly persons with type 2 diabetes mellitus pathology.

The concentration of bacterial plasmalogen 18a and endotoxin in the blood of elderly people 45-90 years old with the pathology of type 2 diabetes mellitus (DM 2) - the main group and without diabetes mellitus - the comparison group was investigated. The concentration of both plasmalogen 18a and endotoxin in the blood of individuals with DM 2 pathology is statistically significantly higher than in the blood of individuals without DM 2 pathology. To assess the state of microbiocenosis and predict type 2 diabetes mellitus, decisive rules have been determined in the form of threshold values of plasma concentrations 18a and endotoxin in the blood of elderly people with a suspected or established diagnosis of type 2 diabetes. Using ROC analysis, it was found that values above 20.66 µg / ml for plasmalogen 18a, and 0.48 nmol / ml for endotoxin, determine the presence of type 2 diabetes mellitus pathology in the 45-90 age group.

Development of test kit for detection of specific IgM to SARS-CoV-2 by immune blotting in the «Line blot¼ format.

Test kit for detection of specific IgM to SARS-CoV-2 by immune blotting in the «Line blot¼ format has been developed. A preliminary study of diagnostic effectivity on clinical samples of blood serum from patients with COVID-19 and healthy donors showed its high sensitivity and specificity. The new test kit allows to detect IgM to all four structural antigens of SARS-CoV-2 and can be used as a confirmatory test to verify indeterminant screening results in laboratory etiological diagnosis of COVID-19.

Development of an enzyme-linked immunosorbent assay for the detection of IgG-antibodies to the causative agent of COVID-19 in human serum (plasma).

A new original Russian test kit for the detection of IgG-antibodies to the causative agent of COVID-19 - coronavirus SARS-CoV-2 by the method of enzyme-linked immunosorbent assay (ELISA) on a solid-phase «ELISA-SARS-CoV-2-AT-G¼ has been developed. In comparative tests with similar test systems «Vitrotest® SARS-CoV-2 IgG¼ (Vitrotest, Ukraine) and «Anti-SARS-Cov-2 ELISA (IgG)¼ (EUROIMMUN AG, Germany) high diagnostic efficiency of the new test system was shown.

Immune blotting as a method for diagnosis of toxoplasmosis.

Test kit for detection of IgG-antibodies to individual antigens of Toxoplasma gondii by immune blotting («Western blot¼ format) has been developed. Laboratory testing with first international WHO standard «Anti-toxoplasma serum (IgG), human, Lyophilized, 20 IU / ampoule¼ (NIBSC, Great Britain) demonstrated the analytical sensitivity of the new kit equal to 10 IU / ml. Study of the diagnostic efficiency of the new kit showed its high sensitivity, equal to 98.51 - 100%, and high specificity, equal to 99.5 - 100%. New test kit is intended for confirmatory testing in laboratory diagnostics of toxoplasmosis.

[Comparative evaluation of the immunochromatographic test for detection of hemoglobin.] / Сравнительная апробация Ð¸Ð¼Ð¼ÑƒÐ½Ð¾Ñ Ñ€Ð¾Ð¼Ð°Ñ‚Ð¾Ð³Ñ€Ð°Ñ„Ð¸Ñ‡ÐµÑÐºÐ¾Ð¹ тест-системы для выявления гемоглобина в кале.

The evaluating results of the diagnostic characteristics of an immunochromatographic test for the detection of fecal occult blood (hemoglobin) were presents in the article. The test was approved samples without hemoglobin and model samples containing of standardized preparation of hemoglobin in various concentrations in comparison with two immunochromatographic test systems. The developed test system identified hemoglobin in concentrations higher 50 ng/ml in 99,2% cases, the specificity of the analysis was 98,1%. The obtained levels of sensitivity and specificity were higher the similar parameters for the compared test systems. The immunochromatographic FOB-test can be used for rapid analysis in the primary screening of colorectal cancer.

[Representation of the results when testing for syphilis.] / Оформление результатов ÑÐºÑ€Ð¸Ð½Ð¸Ð½Ð³Ð¾Ð²Ñ‹Ñ Ð¸ Ð²ÐµÑ€Ð¸Ñ„Ð¸Ñ†Ð¸Ñ€ÑƒÑŽÑ‰Ð¸Ñ Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ð¹ на сифилис.

In medical organizations special forms (approved by orders of the Ministry of health and the Central statistical office) are used to present the results of laboratory examination of the population for syphilis. Currently, these forms include laboratory technologies that do not meet modern standards. In 2018-2019 the normative documents, regulating the use of accounting forms of medical documentation in the public health system, and the practice of using forms with the results of laboratory examination for syphilis were studied in 35 branches of 3 medical organizations of the Lyubertsy district of the Moscow region and in 18 branches of Moscow city was conducted. It is established that in the medical organizations of Moscow and the Moscow region are used the different forms of the registration forms approved by orders of the Ministry of health of the USSR which have now become invalid. The structure of the forms with the results of the population survey for syphilis does not correspond to the applied modern technologies and standards of the survey. On the basis of requirements of modern regulatory documents two new forms of registration forms for the presentation of results of laboratory researches have been developed, within 1,5 years tested and offered for application: one form at screening of the population for syphilis and another - for performing verification examinations for diagnostics and the subsequent clinical and serological supervision.

[Infection with Epstein-Barr virus in certain groups of the population of the Moscow region.]

The serum of 1006 children aged 0-18 years and elderly people aged 60 to 90 years and older for the presence of specific class G immunoglobulins to the Epstein-Barr virus was studied using enzyme immunoassay. The dependence of seropositivity of children on their age and seropositivity of more than 98% of all surveyed elderly people is shown.

[Development of a set of reagents for detection of class G immunoglobulins to human herpes virus type 7 by the method of immune blotting in the format «Western blot¼.]

A new original domestic set of reagents has been developed for the determination of class G immunoglobulins to individual human herpes virus antigens of type 7 by the method of immune blotting in the "Western-blot" format. Preliminary clinical trials were conducted using 134 serums of healthy children aged 1-16 years who underwent diagnostic testing. Stud y of diagnostic efficiency ofthe new kit showed high sensitivity, comparable to the sensitivity of the reaction indirect immunofluorescence and high specificity, which is manifested in the absence of false positive results when testing samples containing immunoglobulin G to herpes virus 6 type.

[Immunochromatographic test for determination of D-dimer.]

The developing and the testing results of an immunochromatographic test for the D-dimer qualitative determination were presents in the article. The test was approved blood plasma samples in comparison with a quantitative enzyme-linked immunosorbent assay (ELISA). The results of assay with developed test were the same with ELISA results for 87,1% and 100% for samples with increased (more than 400 ng/ml FEU) and normal concentration of D-dimer, respectively. The immunochromatographic test for determination of D-dimer can be included in the diagnostic strategy as a cut test after the assessment of venous thromboembolism risk.

[First results of application of an enzyme immunoassay in the serological diagnostics of infection caused by herpesvirus type 7.]

HHV-7 (Human Herpes virus type 7) is a relatively recently discovered, ubiquitous beta herpes virus. In Russia, the diagnosis of HHV-7 infection is carried out by PCR, which determines the DNA in serum or plasma, if there are clinical indications. Laboratory serological tests for HHV-7 are not performed. Currently, in the scientific literature, information on clinical studies of HHV-7 infection, its epidemiology, and its role in the etiopathogenesis of various diseases is insufficient. This article presents the results of the development of a test kit "ELISA-HHV-7-IgG", designed to detect specific IgG to HHV-7 by enzyme-linked immunosorbent assay (ELISA). Studies of the sensitivity and specificity of the developed test kit, including potential cross-reactive samples positive for other herpesvirus infections, were carried out. In the context of serological diagnostics, we studied samples of the sera of children (n = 167) and old people (n = 238) for the presence of IgG to HHV-7 with confirmation of the results in the indirect immunofluorescence reaction and the immune blotting reaction. The research results are evidence of the possibility of using the developed test kit for serological diagnosis of HHV-7 infection.

[Immunochromatographic test for detection of fecal occult blood.]

The developing and evaluating results of the diagnostic characteristics of an immunochromatographic test for the detection of fecal occult blood (hemoglobin) were presents in the article. The test was approved samples without hemoglobin and model samples containing of standardized preparation of hemoglobin in various concentrations. The developed test system identified hemoglobin in concentrations higher 5 µg / g feces in 97.6% cases, the specificity of the analysis was 100%. The immunochromatographic FOB-test can be used for rapid analysis in the primary screening of colorectal cancer.

[Standardization of component manufacturing conditions and setting up the immunofluorescence reaction on the example of syphilis laboratory diagnostics (literature review).]

The use of technology for preparing components for immunofluorescence reaction (RIF) in a number of clinical diagnostic laboratories (CDL) in accordance with the recommendations of the Guidelines introduced by order of the RF Ministry of Health No 87 dated March 26, 2001 does not guarantee the required standardization of RIF preparation and conduct conditions. The latter is achieved by using the appropriate industrial production reagent kits in the СDL practice.

[Current state and development trends of domestic medical device market for in vitro diagnostics in the segment of diagnostic reagents and their kits.]

The problem of import substitution in the Russian medical device market for in vitro diagnostics (MDivd) has not been solved, despite a number of measures taken by the government of the Russian Federation since 2011. The reasons hampering this process are the lack of modern analytical equipment of domestic production and deficiencies of the current regulatory system for MDivd circulation in the market. It is proposed the transition from licensing system of MDivd registration to notification system as a radical measure to solve the problem of import substitution.

[Application of indirect enzyme immunoassay to detect specific IgM for human herpes virus type 6.]

In recent years, timely diagnosis of human herpes virus infection of type 6 (HHV6), especially in pediatrics, has acquired particular relevance. In the spectrum of serological diagnostics, an indirect ELISA method was used to detect IgM against HHV-6 in a study of samples of children sera (198), conditionally healthy patients (148) and donors (145). The use of ELISA IgM HHV6 is more significant for the expansion of the complex of the diagnostic study of the disease associated with primary HHV6 infection or its reactivation.