A broad-range PCR technique for the diagnosis of culture-negative osteomyelitis

Osteomyelitis is a rare disease that is often caused by an infection. In case of microbiology analyses failure, molecular assay seems appropriate for the identification of the pathogen. Broad-range PCR is a popular tool to amplify the gene of 16S ribosomal RNA – the component of the 30S subunit of the bacterial ribosome present in various species. The subsequent sequencing of the amplified gene enables scientists to determine the bacteria species. In this review, we discuss studies and case reports where the osteomyelitis causative agent was revealed by means of broad-range PCR. The purpose of the analysis is to assess the relevance and significance of this method for the diagnosis of osteomyelitis in patients. Numerous successful applications of wide-range PCR followed by sequencing in order to identify the causative agent of osteomyelitis have proven that this method is a useful tool in cases where the culture analysеs showed negative results

Efficient soluble expression and purification of influenza A and B nucleoproteins in E. coli

Viral nucleoprotein (NP) is an abundant essential protein of an influenza virus that has important functional and structural roles. It participates in genomic organization, nuclear trafficking, RNA transcription, and genome replication. From the research point of view, NP is an important protein that is used in the development of new diagnostic methods and vaccination protocols. NP is a promising target for antiviral chemotherapeutic drugs as well. Successful expression of codon-optimized NP genes in E. coli has been reported. In this study, we demonstrated the efficient expression and purification of soluble NPs of influenza A and B viruses in E. coli without the codon-optimization of DNA sequences. This procedure preserves the co-translational protein folding, protein configuration and function. Obtained NPs of influenza A and B viruses were monomers and reacted well with mouse specific antibodies according to Western blot analysis. Our results show that both influenza A and influenza B virus NPs can be efficiently expressed in E. coli without codon-optimization.Viral nucleoprotein (NP) is an abundant essential protein of an influenza virus that has important functional and structural roles. It participates in genomic organization, nuclear trafficking, RNA transcription, and genome replication. From the research point of view, NP is an important protein that is used in the development of new diagnostic methods and vaccination protocols. NP is a promising target for antiviral chemotherapeutic drugs as well. Successful expression of codon-optimized NP genes in E. coli has been reported. In this study, we demonstrated the efficient expression and purification of soluble NPs of influenza A and B viruses in E. coli without the codon-optimization of DNA sequences. This procedure preserves the co-translational protein folding, protein configuration and function. Obtained NPs of influenza A and B viruses were monomers and reacted well with mouse specific antibodies according to Western blot analysis. Our results show that both influenza A and influenza B virus NPs can be efficiently expressed in E. coli without codon-optimization

Случай сочетанной инфекции оспы обезьян и вируса простого герпеса 1 типа

The first Russian clinical case of monkeypox in combination with herpes simplex type 1 infection in a 29-year-old man who returned from Portugal is described. The protocols for sequencing the virus genome are presented. Particular attention is paid to the difficulty of diagnosing vesicular rash in patients with a suspicious history.Описан первый в России клинический случай оспы обезьян в сочетании с инфекцией простого герпеса 1-го типа у мужчины 29 лет, вернувшегося из Испании. Представлены протоколы секвенирования генома вируса. Особое внимание уделено сложности диагностики везикулярной сыпи у пациентов с подозрительным анамнезом

Detection of residual <i>E. coli</i> and CHO<i></i>host-cell<i></i>DNA in recombinant proteins by qPCR

Production of recombinant proteins is a steadily growing industry in Russia and all over the world. Both producer and substance should be properly characterized and tested against all safety criteria. One of the mandatory requirements to the mentioned drugs is the assessment of the content of residual DNA-producing cells, which should be less than 10 ng per dose. There are not many commercial kits for detection of residual DNA and they are based on four different methods. The article provides a brief overview of these methods and highlights their advantages and disadvantages. Common disadvantage of all the commercial kits is their price. The main goal of the research was to choose an optimal method of DNA extraction from protein substances and samples of various protein purification steps, as well as to work over measuring the amount of residual E. coli DNA and CHO by qPCR method not using commercial kits. The article also provides with a number of practical recommendations on specific aspects of DNA extraction and reference standard storage. The aim of the study was to find a reliable and inexpensive method for determining residual DNA-producing cells in recombinant protein samples. The article provides with an overview of DNA extraction methods, stipulates the necessity of DNA extraction prior to the analysis. The advantage was given to the method of spin-column extraction. Optimal DNA extraction method for its assay in a substance is the method, which provides with a stable DNA yield, including different chemical structure of the samples, and upon the condition that no protein and other impurities are detected in the isolated DNA solution. The proposed method for DNA spin-column extraction is the least labor-intensive, is optimized for DNA isolation from protein substances and samples of various protein purification steps. Self preparation of solutions for DNA extraction is a simple procedure and can reduce analysis costs. The report on successful adaptation of the methods of residual E. coli and CHO DNA detection by qPCR using fluorescent probes was provided. The sensitivity of the method was demonstrated at least 1 pg/ml for the analysis of the amount of residual DNA both for E. coli and CHO

Possibilities of qPCR control of mycoplasma contamination of cell cultures

Mycoplasmas are the main contaminants of cells cultures. They persist in cell cultures and can extensively affect host cell functions. Conducting experiments or producing protein in contaminated cultures are impractical. The aim of the study was to explore the possibility of quick detection of mycoplasma contamination of cell cultures and biotechnological products by a previously developed method which was modified to make it simpler and more affordable in Russia; and to assess the possibility of method validation for quality control. The authors chose the most applicable qPCR method of all qPCR methods currently used for mycoplasma detection, and modified it in the following way: expensive MGB-probes which cannot be synthesized in Russia were substituted by ordinary fluorescence probes. The reproducibility and sensitivity of the modified method were tested with M. hominis. The sensitivity of the test was equal to 10 mycoplasma gene copies per reaction. Comparison of the obtained results with regulatory requirements for mycoplasma detection showed that the proposed method complies with current official requirements and could be used as the main method for routine prompt cell culture testing for mycoplasma contamination

Определение остаточной ДНК клеток-продуцентов E. coli и CHO в субстанциях рекомбинантых белков методом qPCR

Production of recombinant proteins is a steadily growing industry in Russia and all over the world. Both producer and substance should be properly characterized and tested against all safety criteria. One of the mandatory requirements to the mentioned drugs is the assessment of the content of residual DNA-producing cells, which should be less than 10 ng per dose. There are not many commercial kits for detection of residual DNA and they are based on four different methods. The article provides a brief overview of these methods and highlights their advantages and disadvantages. Common disadvantage of all the commercial kits is their price. The main goal of the research was to choose an optimal method of DNA extraction from protein substances and samples of various protein purification steps, as well as to work over measuring the amount of residual E. coli DNA and CHO by qPCR method not using commercial kits. The article also provides with a number of practical recommendations on specific aspects of DNA extraction and reference standard storage. The aim of the study was to find a reliable and inexpensive method for determining residual DNA-producing cells in recombinant protein samples. The article provides with an overview of DNA extraction methods, stipulates the necessity of DNA extraction prior to the analysis. The advantage was given to the method of spin-column extraction. Optimal DNA extraction method for its assay in a substance is the method, which provides with a stable DNA yield, including different chemical structure of the samples, and upon the condition that no protein and other impurities are detected in the isolated DNA solution. The proposed method for DNA spin-column extraction is the least labor-intensive, is optimized for DNA isolation from protein substances and samples of various protein purification steps. Self preparation of solutions for DNA extraction is a simple procedure and can reduce analysis costs. The report on successful adaptation of the methods of residual E. coli and CHO DNA detection by qPCR using fluorescent probes was provided. The sensitivity of the method was demonstrated at least 1 pg/ml for the analysis of the amount of residual DNA both for E. coli and CHO.Рынок препаратов рекомбинантых белков растет и развивается во всем мире, в том числе и в России. Как продуцент, так и субстанция, должны быть тщательно охарактеризованы и проверены по всем критериям безопасности. Среди обязательных требований, предъявляемых к таким препаратам - содержание остаточной ДНК клеток-продуцентов должно быть не более 10 нг на дозу. Коммерческих наборов для определения остаточной ДНК в мире относительно немного, они основаны на четырех разных методах. В статье представлен краткий обзор этих методов, обозначены их преимущества и недостатки. Общим недостатком всех коммерческих наборов является стоимость. Основными задачами работы было подобрать оптимальный метод выделения ДНК из белковых субстанций и образцов различных этапов очистки белка, отработать определение количества остаточной ДНК Escherichia coli и клеток яичника китайского хомяка (CHO) методом количественной полимеразной цепной реакции (qPCR) без использования коммерческих наборов. Статья также содержит ряд практических рекомендаций по особенностям выделения ДНК и хранению стандарта. Целью исследования был поиск достоверного и недорогого метода определения содержания остаточной ДНК клеток-продуцентов в образцах рекомбинантых белков. В статье приводится обзор методов выделения ДНК для анализа, обоснована необходимость выделения ДНК перед анализом, преимущество отдано методу выделения на спин-колонках. Оптимальный метод выделения ДНК для определения ее количества в субстанции такой, при котором выход ДНК стабилен, в том числе и при различных химических составах исследуемых образцов, а в растворе выделенной ДНК отсутствует белок и другие примеси. Предложенный метод выделения ДНК на спин-колонках является наименее трудозатратным, оптимизирован для выделения ДНК из белковых субстанций и образцов различных стадий очистки белков. Самостоятельное приготовление растворов для выделения ДНК является простой процедурой и может снизить затраты на анализ. Представлен отчет об успешной адаптации методик определения остаточной ДНК E. coli и CHO методом qPCR с использованием флуоресцентных зондов. Продемонстрирована чувствительность метода не менее 1 пг/мл как при анализе количества остаточной ДНК E. coli, так и CHO

Изучение возможности использования метода qPCR для контроля отсутствия микоплазменной контаминации в клеточных культурах

Mycoplasmas are the main contaminants of cells cultures. They persist in cell cultures and can extensively affect host cell functions. Conducting experiments or producing protein in contaminated cultures are impractical. The aim of the study was to explore the possibility of quick detection of mycoplasma contamination of cell cultures and biotechnological products by a previously developed method which was modified to make it simpler and more affordable in Russia; and to assess the possibility of method validation for quality control. The authors chose the most applicable qPCR method of all qPCR methods currently used for mycoplasma detection, and modified it in the following way: expensive MGB-probes which cannot be synthesized in Russia were substituted by ordinary fluorescence probes. The reproducibility and sensitivity of the modified method were tested with M. hominis. The sensitivity of the test was equal to 10 mycoplasma gene copies per reaction. Comparison of the obtained results with regulatory requirements for mycoplasma detection showed that the proposed method complies with current official requirements and could be used as the main method for routine prompt cell culture testing for mycoplasma contamination.Микоплазмы - основные контаминанты клеточных культур. Персистируя в клеточных культурах, микоплазмы многообразно влияют на функционирование клеток-хозяев. Проведение экспериментов на таких контаминированных клетках нерационально, как и наработка в них белка. Цель работы - изучить возможность быстрого выявления контаминации микоплазмами культур клеток и биотехнологических продуктов с воспроизведением ранее разработанной методики, при внесении в нее упрощающих модификаций, повышающих доступностность такого анализа в России, а также оценить возможность валидации методики для контроля в процессе производства. Из уже существующих методик детекции микоплазмы методом qPCR (quantitative PCR) была выбрана наиболее пригодная для работы, произведена модификация анализа: дорогостоящие и недоступные к синтезу в России MGB-зонды были заменены на обычные флуоресцентные. Воспроизводимость и чувствительность модифицированной методики были изучены на примере работы с M. hominis. Чувствительность данного теста достигает 10 генных копий микоплазм на реакцию. На основании сопоставления полученных результатов и регламентируемых нормативными документами требований к методам детекции микоплазм сделан вывод, что предложенная методика на основе qPCR анализа хорошо подходит для своевременной и регулярной проверки культур клеток на предмет контаминации микоплазмами, теоретически нет никаких противоречий с современной регламентирующей документацией для использования этого метода в качестве основного