7 research outputs found
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 β 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 analysΠ΅s 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
Π‘Π»ΡΡΠ°ΠΉ ΡΠΎΡΠ΅ΡΠ°Π½Π½ΠΎΠΉ ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠΈ ΠΎΡΠΏΡ ΠΎΠ±Π΅Π·ΡΡΠ½ ΠΈ Π²ΠΈΡΡΡΠ° ΠΏΡΠΎΡΡΠΎΠ³ΠΎ Π³Π΅ΡΠΏΠ΅ΡΠ° 1 ΡΠΈΠΏΠ°
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.ΠΠΏΠΈΡΠ°Π½ ΠΏΠ΅ΡΠ²ΡΠΉ Π² Π ΠΎΡΡΠΈΠΈ ΠΊΠ»ΠΈΠ½ΠΈΡΠ΅ΡΠΊΠΈΠΉ ΡΠ»ΡΡΠ°ΠΉ ΠΎΡΠΏΡ ΠΎΠ±Π΅Π·ΡΡΠ½ Π² ΡΠΎΡΠ΅ΡΠ°Π½ΠΈΠΈ Ρ ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠ΅ΠΉ ΠΏΡΠΎΡΡΠΎΠ³ΠΎ Π³Π΅ΡΠΏΠ΅ΡΠ° 1-Π³ΠΎ ΡΠΈΠΏΠ° Ρ ΠΌΡΠΆΡΠΈΠ½Ρ 29 Π»Π΅Ρ, Π²Π΅ΡΠ½ΡΠ²ΡΠ΅Π³ΠΎΡΡ ΠΈΠ· ΠΡΠΏΠ°Π½ΠΈΠΈ. ΠΡΠ΅Π΄ΡΡΠ°Π²Π»Π΅Π½Ρ ΠΏΡΠΎΡΠΎΠΊΠΎΠ»Ρ ΡΠ΅ΠΊΠ²Π΅Π½ΠΈΡΠΎΠ²Π°Π½ΠΈΡ Π³Π΅Π½ΠΎΠΌΠ° Π²ΠΈΡΡΡΠ°. ΠΡΠΎΠ±ΠΎΠ΅ Π²Π½ΠΈΠΌΠ°Π½ΠΈΠ΅ ΡΠ΄Π΅Π»Π΅Π½ΠΎ ΡΠ»ΠΎΠΆΠ½ΠΎΡΡΠΈ Π΄ΠΈΠ°Π³Π½ΠΎΡΡΠΈΠΊΠΈ Π²Π΅Π·ΠΈΠΊΡΠ»ΡΡΠ½ΠΎΠΉ ΡΡΠΏΠΈ Ρ ΠΏΠ°ΡΠΈΠ΅Π½ΡΠΎΠ² Ρ ΠΏΠΎΠ΄ΠΎΠ·ΡΠΈΡΠ΅Π»ΡΠ½ΡΠΌ Π°Π½Π°ΠΌΠ½Π΅Π·ΠΎΠΌ
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
ΠΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΠ΅ ΠΎΡΡΠ°ΡΠΎΡΠ½ΠΎΠΉ ΠΠΠ ΠΊΠ»Π΅ΡΠΎΠΊ-ΠΏΡΠΎΠ΄ΡΡΠ΅Π½ΡΠΎΠ² E. coli ΠΈ CHO Π² ΡΡΠ±ΡΡΠ°Π½ΡΠΈΡΡ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΡΡ Π±Π΅Π»ΠΊΠΎΠ² ΠΌΠ΅ΡΠΎΠ΄ΠΎΠΌ 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.Π ΡΠ½ΠΎΠΊ ΠΏΡΠ΅ΠΏΠ°ΡΠ°ΡΠΎΠ² ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΡΡ
Π±Π΅Π»ΠΊΠΎΠ² ΡΠ°ΡΡΠ΅Ρ ΠΈ ΡΠ°Π·Π²ΠΈΠ²Π°Π΅ΡΡΡ Π²ΠΎ Π²ΡΠ΅ΠΌ ΠΌΠΈΡΠ΅, Π² ΡΠΎΠΌ ΡΠΈΡΠ»Π΅ ΠΈ Π² Π ΠΎΡΡΠΈΠΈ. ΠΠ°ΠΊ ΠΏΡΠΎΠ΄ΡΡΠ΅Π½Ρ, ΡΠ°ΠΊ ΠΈ ΡΡΠ±ΡΡΠ°Π½ΡΠΈΡ, Π΄ΠΎΠ»ΠΆΠ½Ρ Π±ΡΡΡ ΡΡΠ°ΡΠ΅Π»ΡΠ½ΠΎ ΠΎΡ
Π°ΡΠ°ΠΊΡΠ΅ΡΠΈΠ·ΠΎΠ²Π°Π½Ρ ΠΈ ΠΏΡΠΎΠ²Π΅ΡΠ΅Π½Ρ ΠΏΠΎ Π²ΡΠ΅ΠΌ ΠΊΡΠΈΡΠ΅ΡΠΈΡΠΌ Π±Π΅Π·ΠΎΠΏΠ°ΡΠ½ΠΎΡΡΠΈ. Π‘ΡΠ΅Π΄ΠΈ ΠΎΠ±ΡΠ·Π°ΡΠ΅Π»ΡΠ½ΡΡ
ΡΡΠ΅Π±ΠΎΠ²Π°Π½ΠΈΠΉ, ΠΏΡΠ΅Π΄ΡΡΠ²Π»ΡΠ΅ΠΌΡΡ
ΠΊ ΡΠ°ΠΊΠΈΠΌ ΠΏΡΠ΅ΠΏΠ°ΡΠ°ΡΠ°ΠΌ - ΡΠΎΠ΄Π΅ΡΠΆΠ°Π½ΠΈΠ΅ ΠΎΡΡΠ°ΡΠΎΡΠ½ΠΎΠΉ ΠΠΠ ΠΊΠ»Π΅ΡΠΎΠΊ-ΠΏΡΠΎΠ΄ΡΡΠ΅Π½ΡΠΎΠ² Π΄ΠΎΠ»ΠΆΠ½ΠΎ Π±ΡΡΡ Π½Π΅ Π±ΠΎΠ»Π΅Π΅ 10 Π½Π³ Π½Π° Π΄ΠΎΠ·Ρ. ΠΠΎΠΌΠΌΠ΅ΡΡΠ΅ΡΠΊΠΈΡ
Π½Π°Π±ΠΎΡΠΎΠ² Π΄Π»Ρ ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΡ ΠΎΡΡΠ°ΡΠΎΡΠ½ΠΎΠΉ ΠΠΠ Π² ΠΌΠΈΡΠ΅ ΠΎΡΠ½ΠΎΡΠΈΡΠ΅Π»ΡΠ½ΠΎ Π½Π΅ΠΌΠ½ΠΎΠ³ΠΎ, ΠΎΠ½ΠΈ ΠΎΡΠ½ΠΎΠ²Π°Π½Ρ Π½Π° ΡΠ΅ΡΡΡΠ΅Ρ
ΡΠ°Π·Π½ΡΡ
ΠΌΠ΅ΡΠΎΠ΄Π°Ρ
. Π ΡΡΠ°ΡΡΠ΅ ΠΏΡΠ΅Π΄ΡΡΠ°Π²Π»Π΅Π½ ΠΊΡΠ°ΡΠΊΠΈΠΉ ΠΎΠ±Π·ΠΎΡ ΡΡΠΈΡ
ΠΌΠ΅ΡΠΎΠ΄ΠΎΠ², ΠΎΠ±ΠΎΠ·Π½Π°ΡΠ΅Π½Ρ ΠΈΡ
ΠΏΡΠ΅ΠΈΠΌΡΡΠ΅ΡΡΠ²Π° ΠΈ Π½Π΅Π΄ΠΎΡΡΠ°ΡΠΊΠΈ. ΠΠ±ΡΠΈΠΌ Π½Π΅Π΄ΠΎΡΡΠ°ΡΠΊΠΎΠΌ Π²ΡΠ΅Ρ
ΠΊΠΎΠΌΠΌΠ΅ΡΡΠ΅ΡΠΊΠΈΡ
Π½Π°Π±ΠΎΡΠΎΠ² ΡΠ²Π»ΡΠ΅ΡΡΡ ΡΡΠΎΠΈΠΌΠΎΡΡΡ. ΠΡΠ½ΠΎΠ²Π½ΡΠΌΠΈ Π·Π°Π΄Π°ΡΠ°ΠΌΠΈ ΡΠ°Π±ΠΎΡΡ Π±ΡΠ»ΠΎ ΠΏΠΎΠ΄ΠΎΠ±ΡΠ°ΡΡ ΠΎΠΏΡΠΈΠΌΠ°Π»ΡΠ½ΡΠΉ ΠΌΠ΅ΡΠΎΠ΄ Π²ΡΠ΄Π΅Π»Π΅Π½ΠΈΡ ΠΠΠ ΠΈΠ· Π±Π΅Π»ΠΊΠΎΠ²ΡΡ
ΡΡΠ±ΡΡΠ°Π½ΡΠΈΠΉ ΠΈ ΠΎΠ±ΡΠ°Π·ΡΠΎΠ² ΡΠ°Π·Π»ΠΈΡΠ½ΡΡ
ΡΡΠ°ΠΏΠΎΠ² ΠΎΡΠΈΡΡΠΊΠΈ Π±Π΅Π»ΠΊΠ°, ΠΎΡΡΠ°Π±ΠΎΡΠ°ΡΡ ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΠ΅ ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π° ΠΎΡΡΠ°ΡΠΎΡΠ½ΠΎΠΉ ΠΠΠ Escherichia coli ΠΈ ΠΊΠ»Π΅ΡΠΎΠΊ ΡΠΈΡΠ½ΠΈΠΊΠ° ΠΊΠΈΡΠ°ΠΉΡΠΊΠΎΠ³ΠΎ Ρ
ΠΎΠΌΡΠΊΠ° (CHO) ΠΌΠ΅ΡΠΎΠ΄ΠΎΠΌ ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΎΠΉ ΠΏΠΎΠ»ΠΈΠΌΠ΅ΡΠ°Π·Π½ΠΎΠΉ ΡΠ΅ΠΏΠ½ΠΎΠΉ ΡΠ΅Π°ΠΊΡΠΈΠΈ (qPCR) Π±Π΅Π· ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ΠΈΡ ΠΊΠΎΠΌΠΌΠ΅ΡΡΠ΅ΡΠΊΠΈΡ
Π½Π°Π±ΠΎΡΠΎΠ². Π‘ΡΠ°ΡΡΡ ΡΠ°ΠΊΠΆΠ΅ ΡΠΎΠ΄Π΅ΡΠΆΠΈΡ ΡΡΠ΄ ΠΏΡΠ°ΠΊΡΠΈΡΠ΅ΡΠΊΠΈΡ
ΡΠ΅ΠΊΠΎΠΌΠ΅Π½Π΄Π°ΡΠΈΠΉ ΠΏΠΎ ΠΎΡΠΎΠ±Π΅Π½Π½ΠΎΡΡΡΠΌ Π²ΡΠ΄Π΅Π»Π΅Π½ΠΈΡ ΠΠΠ ΠΈ Ρ
ΡΠ°Π½Π΅Π½ΠΈΡ ΡΡΠ°Π½Π΄Π°ΡΡΠ°. Π¦Π΅Π»ΡΡ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΡ Π±ΡΠ» ΠΏΠΎΠΈΡΠΊ Π΄ΠΎΡΡΠΎΠ²Π΅ΡΠ½ΠΎΠ³ΠΎ ΠΈ Π½Π΅Π΄ΠΎΡΠΎΠ³ΠΎΠ³ΠΎ ΠΌΠ΅ΡΠΎΠ΄Π° ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΡ ΡΠΎΠ΄Π΅ΡΠΆΠ°Π½ΠΈΡ ΠΎΡΡΠ°ΡΠΎΡΠ½ΠΎΠΉ ΠΠΠ ΠΊΠ»Π΅ΡΠΎΠΊ-ΠΏΡΠΎΠ΄ΡΡΠ΅Π½ΡΠΎΠ² Π² ΠΎΠ±ΡΠ°Π·ΡΠ°Ρ
ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΡΡ
Π±Π΅Π»ΠΊΠΎΠ². Π ΡΡΠ°ΡΡΠ΅ ΠΏΡΠΈΠ²ΠΎΠ΄ΠΈΡΡΡ ΠΎΠ±Π·ΠΎΡ ΠΌΠ΅ΡΠΎΠ΄ΠΎΠ² Π²ΡΠ΄Π΅Π»Π΅Π½ΠΈΡ ΠΠΠ Π΄Π»Ρ Π°Π½Π°Π»ΠΈΠ·Π°, ΠΎΠ±ΠΎΡΠ½ΠΎΠ²Π°Π½Π° Π½Π΅ΠΎΠ±Ρ
ΠΎΠ΄ΠΈΠΌΠΎΡΡΡ Π²ΡΠ΄Π΅Π»Π΅Π½ΠΈΡ ΠΠΠ ΠΏΠ΅ΡΠ΅Π΄ Π°Π½Π°Π»ΠΈΠ·ΠΎΠΌ, ΠΏΡΠ΅ΠΈΠΌΡΡΠ΅ΡΡΠ²ΠΎ ΠΎΡΠ΄Π°Π½ΠΎ ΠΌΠ΅ΡΠΎΠ΄Ρ Π²ΡΠ΄Π΅Π»Π΅Π½ΠΈΡ Π½Π° ΡΠΏΠΈΠ½-ΠΊΠΎΠ»ΠΎΠ½ΠΊΠ°Ρ
. ΠΠΏΡΠΈΠΌΠ°Π»ΡΠ½ΡΠΉ ΠΌΠ΅ΡΠΎΠ΄ Π²ΡΠ΄Π΅Π»Π΅Π½ΠΈΡ ΠΠΠ Π΄Π»Ρ ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΡ Π΅Π΅ ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π° Π² ΡΡΠ±ΡΡΠ°Π½ΡΠΈΠΈ ΡΠ°ΠΊΠΎΠΉ, ΠΏΡΠΈ ΠΊΠΎΡΠΎΡΠΎΠΌ Π²ΡΡ
ΠΎΠ΄ ΠΠΠ ΡΡΠ°Π±ΠΈΠ»Π΅Π½, Π² ΡΠΎΠΌ ΡΠΈΡΠ»Π΅ ΠΈ ΠΏΡΠΈ ΡΠ°Π·Π»ΠΈΡΠ½ΡΡ
Ρ
ΠΈΠΌΠΈΡΠ΅ΡΠΊΠΈΡ
ΡΠΎΡΡΠ°Π²Π°Ρ
ΠΈΡΡΠ»Π΅Π΄ΡΠ΅ΠΌΡΡ
ΠΎΠ±ΡΠ°Π·ΡΠΎΠ², Π° Π² ΡΠ°ΡΡΠ²ΠΎΡΠ΅ Π²ΡΠ΄Π΅Π»Π΅Π½Π½ΠΎΠΉ ΠΠΠ ΠΎΡΡΡΡΡΡΠ²ΡΠ΅Ρ Π±Π΅Π»ΠΎΠΊ ΠΈ Π΄ΡΡΠ³ΠΈΠ΅ ΠΏΡΠΈΠΌΠ΅ΡΠΈ. ΠΡΠ΅Π΄Π»ΠΎΠΆΠ΅Π½Π½ΡΠΉ ΠΌΠ΅ΡΠΎΠ΄ Π²ΡΠ΄Π΅Π»Π΅Π½ΠΈΡ ΠΠΠ Π½Π° ΡΠΏΠΈΠ½-ΠΊΠΎΠ»ΠΎΠ½ΠΊΠ°Ρ
ΡΠ²Π»ΡΠ΅ΡΡΡ Π½Π°ΠΈΠΌΠ΅Π½Π΅Π΅ ΡΡΡΠ΄ΠΎΠ·Π°ΡΡΠ°ΡΠ½ΡΠΌ, ΠΎΠΏΡΠΈΠΌΠΈΠ·ΠΈΡΠΎΠ²Π°Π½ Π΄Π»Ρ Π²ΡΠ΄Π΅Π»Π΅Π½ΠΈΡ ΠΠΠ ΠΈΠ· Π±Π΅Π»ΠΊΠΎΠ²ΡΡ
ΡΡΠ±ΡΡΠ°Π½ΡΠΈΠΉ ΠΈ ΠΎΠ±ΡΠ°Π·ΡΠΎΠ² ΡΠ°Π·Π»ΠΈΡΠ½ΡΡ
ΡΡΠ°Π΄ΠΈΠΉ ΠΎΡΠΈΡΡΠΊΠΈ Π±Π΅Π»ΠΊΠΎΠ². Π‘Π°ΠΌΠΎΡΡΠΎΡΡΠ΅Π»ΡΠ½ΠΎΠ΅ ΠΏΡΠΈΠ³ΠΎΡΠΎΠ²Π»Π΅Π½ΠΈΠ΅ ΡΠ°ΡΡΠ²ΠΎΡΠΎΠ² Π΄Π»Ρ Π²ΡΠ΄Π΅Π»Π΅Π½ΠΈΡ ΠΠΠ ΡΠ²Π»ΡΠ΅ΡΡΡ ΠΏΡΠΎΡΡΠΎΠΉ ΠΏΡΠΎΡΠ΅Π΄ΡΡΠΎΠΉ ΠΈ ΠΌΠΎΠΆΠ΅Ρ ΡΠ½ΠΈΠ·ΠΈΡΡ Π·Π°ΡΡΠ°ΡΡ Π½Π° Π°Π½Π°Π»ΠΈΠ·. ΠΡΠ΅Π΄ΡΡΠ°Π²Π»Π΅Π½ ΠΎΡΡΠ΅Ρ ΠΎΠ± ΡΡΠΏΠ΅ΡΠ½ΠΎΠΉ Π°Π΄Π°ΠΏΡΠ°ΡΠΈΠΈ ΠΌΠ΅ΡΠΎΠ΄ΠΈΠΊ ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ΠΈΡ ΠΎΡΡΠ°ΡΠΎΡΠ½ΠΎΠΉ ΠΠΠ E. coli ΠΈ CHO ΠΌΠ΅ΡΠΎΠ΄ΠΎΠΌ qPCR Ρ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ΠΈΠ΅ΠΌ ΡΠ»ΡΠΎΡΠ΅ΡΡΠ΅Π½ΡΠ½ΡΡ
Π·ΠΎΠ½Π΄ΠΎΠ². ΠΡΠΎΠ΄Π΅ΠΌΠΎΠ½ΡΡΡΠΈΡΠΎΠ²Π°Π½Π° ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΡ ΠΌΠ΅ΡΠΎΠ΄Π° Π½Π΅ ΠΌΠ΅Π½Π΅Π΅ 1 ΠΏΠ³/ΠΌΠ» ΠΊΠ°ΠΊ ΠΏΡΠΈ Π°Π½Π°Π»ΠΈΠ·Π΅ ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²Π° ΠΎΡΡΠ°ΡΠΎΡΠ½ΠΎΠΉ ΠΠΠ E. coli, ΡΠ°ΠΊ ΠΈ CHO
ΠΠ·ΡΡΠ΅Π½ΠΈΠ΅ Π²ΠΎΠ·ΠΌΠΎΠΆΠ½ΠΎΡΡΠΈ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ΠΈΡ ΠΌΠ΅ΡΠΎΠ΄Π° qPCR Π΄Π»Ρ ΠΊΠΎΠ½ΡΡΠΎΠ»Ρ ΠΎΡΡΡΡΡΡΠ²ΠΈΡ ΠΌΠΈΠΊΠΎΠΏΠ»Π°Π·ΠΌΠ΅Π½Π½ΠΎΠΉ ΠΊΠΎΠ½ΡΠ°ΠΌΠΈΠ½Π°ΡΠΈΠΈ Π² ΠΊΠ»Π΅ΡΠΎΡΠ½ΡΡ ΠΊΡΠ»ΡΡΡΡΠ°Ρ
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.ΠΠΈΠΊΠΎΠΏΠ»Π°Π·ΠΌΡ - ΠΎΡΠ½ΠΎΠ²Π½ΡΠ΅ ΠΊΠΎΠ½ΡΠ°ΠΌΠΈΠ½Π°Π½ΡΡ ΠΊΠ»Π΅ΡΠΎΡΠ½ΡΡ
ΠΊΡΠ»ΡΡΡΡ. ΠΠ΅ΡΡΠΈΡΡΠΈΡΡΡ Π² ΠΊΠ»Π΅ΡΠΎΡΠ½ΡΡ
ΠΊΡΠ»ΡΡΡΡΠ°Ρ
, ΠΌΠΈΠΊΠΎΠΏΠ»Π°Π·ΠΌΡ ΠΌΠ½ΠΎΠ³ΠΎΠΎΠ±ΡΠ°Π·Π½ΠΎ Π²Π»ΠΈΡΡΡ Π½Π° ΡΡΠ½ΠΊΡΠΈΠΎΠ½ΠΈΡΠΎΠ²Π°Π½ΠΈΠ΅ ΠΊΠ»Π΅ΡΠΎΠΊ-Ρ
ΠΎΠ·ΡΠ΅Π². ΠΡΠΎΠ²Π΅Π΄Π΅Π½ΠΈΠ΅ ΡΠΊΡΠΏΠ΅ΡΠΈΠΌΠ΅Π½ΡΠΎΠ² Π½Π° ΡΠ°ΠΊΠΈΡ
ΠΊΠΎΠ½ΡΠ°ΠΌΠΈΠ½ΠΈΡΠΎΠ²Π°Π½Π½ΡΡ
ΠΊΠ»Π΅ΡΠΊΠ°Ρ
Π½Π΅ΡΠ°ΡΠΈΠΎΠ½Π°Π»ΡΠ½ΠΎ, ΠΊΠ°ΠΊ ΠΈ Π½Π°ΡΠ°Π±ΠΎΡΠΊΠ° Π² Π½ΠΈΡ
Π±Π΅Π»ΠΊΠ°. Π¦Π΅Π»Ρ ΡΠ°Π±ΠΎΡΡ - ΠΈΠ·ΡΡΠΈΡΡ Π²ΠΎΠ·ΠΌΠΎΠΆΠ½ΠΎΡΡΡ Π±ΡΡΡΡΠΎΠ³ΠΎ Π²ΡΡΠ²Π»Π΅Π½ΠΈΡ ΠΊΠΎΠ½ΡΠ°ΠΌΠΈΠ½Π°ΡΠΈΠΈ ΠΌΠΈΠΊΠΎΠΏΠ»Π°Π·ΠΌΠ°ΠΌΠΈ ΠΊΡΠ»ΡΡΡΡ ΠΊΠ»Π΅ΡΠΎΠΊ ΠΈ Π±ΠΈΠΎΡΠ΅Ρ
Π½ΠΎΠ»ΠΎΠ³ΠΈΡΠ΅ΡΠΊΠΈΡ
ΠΏΡΠΎΠ΄ΡΠΊΡΠΎΠ² Ρ Π²ΠΎΡΠΏΡΠΎΠΈΠ·Π²Π΅Π΄Π΅Π½ΠΈΠ΅ΠΌ ΡΠ°Π½Π΅Π΅ ΡΠ°Π·ΡΠ°Π±ΠΎΡΠ°Π½Π½ΠΎΠΉ ΠΌΠ΅ΡΠΎΠ΄ΠΈΠΊΠΈ, ΠΏΡΠΈ Π²Π½Π΅ΡΠ΅Π½ΠΈΠΈ Π² Π½Π΅Π΅ ΡΠΏΡΠΎΡΠ°ΡΡΠΈΡ
ΠΌΠΎΠ΄ΠΈΡΠΈΠΊΠ°ΡΠΈΠΉ, ΠΏΠΎΠ²ΡΡΠ°ΡΡΠΈΡ
Π΄ΠΎΡΡΡΠΏΠ½ΠΎΡΡΠ½ΠΎΡΡΡ ΡΠ°ΠΊΠΎΠ³ΠΎ Π°Π½Π°Π»ΠΈΠ·Π° Π² Π ΠΎΡΡΠΈΠΈ, Π° ΡΠ°ΠΊΠΆΠ΅ ΠΎΡΠ΅Π½ΠΈΡΡ Π²ΠΎΠ·ΠΌΠΎΠΆΠ½ΠΎΡΡΡ Π²Π°Π»ΠΈΠ΄Π°ΡΠΈΠΈ ΠΌΠ΅ΡΠΎΠ΄ΠΈΠΊΠΈ Π΄Π»Ρ ΠΊΠΎΠ½ΡΡΠΎΠ»Ρ Π² ΠΏΡΠΎΡΠ΅ΡΡΠ΅ ΠΏΡΠΎΠΈΠ·Π²ΠΎΠ΄ΡΡΠ²Π°. ΠΠ· ΡΠΆΠ΅ ΡΡΡΠ΅ΡΡΠ²ΡΡΡΠΈΡ
ΠΌΠ΅ΡΠΎΠ΄ΠΈΠΊ Π΄Π΅ΡΠ΅ΠΊΡΠΈΠΈ ΠΌΠΈΠΊΠΎΠΏΠ»Π°Π·ΠΌΡ ΠΌΠ΅ΡΠΎΠ΄ΠΎΠΌ qPCR (quantitative PCR) Π±ΡΠ»Π° Π²ΡΠ±ΡΠ°Π½Π° Π½Π°ΠΈΠ±ΠΎΠ»Π΅Π΅ ΠΏΡΠΈΠ³ΠΎΠ΄Π½Π°Ρ Π΄Π»Ρ ΡΠ°Π±ΠΎΡΡ, ΠΏΡΠΎΠΈΠ·Π²Π΅Π΄Π΅Π½Π° ΠΌΠΎΠ΄ΠΈΡΠΈΠΊΠ°ΡΠΈΡ Π°Π½Π°Π»ΠΈΠ·Π°: Π΄ΠΎΡΠΎΠ³ΠΎΡΡΠΎΡΡΠΈΠ΅ ΠΈ Π½Π΅Π΄ΠΎΡΡΡΠΏΠ½ΡΠ΅ ΠΊ ΡΠΈΠ½ΡΠ΅Π·Ρ Π² Π ΠΎΡΡΠΈΠΈ MGB-Π·ΠΎΠ½Π΄Ρ Π±ΡΠ»ΠΈ Π·Π°ΠΌΠ΅Π½Π΅Π½Ρ Π½Π° ΠΎΠ±ΡΡΠ½ΡΠ΅ ΡΠ»ΡΠΎΡΠ΅ΡΡΠ΅Π½ΡΠ½ΡΠ΅. ΠΠΎΡΠΏΡΠΎΠΈΠ·Π²ΠΎΠ΄ΠΈΠΌΠΎΡΡΡ ΠΈ ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΡ ΠΌΠΎΠ΄ΠΈΡΠΈΡΠΈΡΠΎΠ²Π°Π½Π½ΠΎΠΉ ΠΌΠ΅ΡΠΎΠ΄ΠΈΠΊΠΈ Π±ΡΠ»ΠΈ ΠΈΠ·ΡΡΠ΅Π½Ρ Π½Π° ΠΏΡΠΈΠΌΠ΅ΡΠ΅ ΡΠ°Π±ΠΎΡΡ Ρ M. hominis. Π§ΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΡ Π΄Π°Π½Π½ΠΎΠ³ΠΎ ΡΠ΅ΡΡΠ° Π΄ΠΎΡΡΠΈΠ³Π°Π΅Ρ 10 Π³Π΅Π½Π½ΡΡ
ΠΊΠΎΠΏΠΈΠΉ ΠΌΠΈΠΊΠΎΠΏΠ»Π°Π·ΠΌ Π½Π° ΡΠ΅Π°ΠΊΡΠΈΡ. ΠΠ° ΠΎΡΠ½ΠΎΠ²Π°Π½ΠΈΠΈ ΡΠΎΠΏΠΎΡΡΠ°Π²Π»Π΅Π½ΠΈΡ ΠΏΠΎΠ»ΡΡΠ΅Π½Π½ΡΡ
ΡΠ΅Π·ΡΠ»ΡΡΠ°ΡΠΎΠ² ΠΈ ΡΠ΅Π³Π»Π°ΠΌΠ΅Π½ΡΠΈΡΡΠ΅ΠΌΡΡ
Π½ΠΎΡΠΌΠ°ΡΠΈΠ²Π½ΡΠΌΠΈ Π΄ΠΎΠΊΡΠΌΠ΅Π½ΡΠ°ΠΌΠΈ ΡΡΠ΅Π±ΠΎΠ²Π°Π½ΠΈΠΉ ΠΊ ΠΌΠ΅ΡΠΎΠ΄Π°ΠΌ Π΄Π΅ΡΠ΅ΠΊΡΠΈΠΈ ΠΌΠΈΠΊΠΎΠΏΠ»Π°Π·ΠΌ ΡΠ΄Π΅Π»Π°Π½ Π²ΡΠ²ΠΎΠ΄, ΡΡΠΎ ΠΏΡΠ΅Π΄Π»ΠΎΠΆΠ΅Π½Π½Π°Ρ ΠΌΠ΅ΡΠΎΠ΄ΠΈΠΊΠ° Π½Π° ΠΎΡΠ½ΠΎΠ²Π΅ qPCR Π°Π½Π°Π»ΠΈΠ·Π° Ρ
ΠΎΡΠΎΡΠΎ ΠΏΠΎΠ΄Ρ
ΠΎΠ΄ΠΈΡ Π΄Π»Ρ ΡΠ²ΠΎΠ΅Π²ΡΠ΅ΠΌΠ΅Π½Π½ΠΎΠΉ ΠΈ ΡΠ΅Π³ΡΠ»ΡΡΠ½ΠΎΠΉ ΠΏΡΠΎΠ²Π΅ΡΠΊΠΈ ΠΊΡΠ»ΡΡΡΡ ΠΊΠ»Π΅ΡΠΎΠΊ Π½Π° ΠΏΡΠ΅Π΄ΠΌΠ΅Ρ ΠΊΠΎΠ½ΡΠ°ΠΌΠΈΠ½Π°ΡΠΈΠΈ ΠΌΠΈΠΊΠΎΠΏΠ»Π°Π·ΠΌΠ°ΠΌΠΈ, ΡΠ΅ΠΎΡΠ΅ΡΠΈΡΠ΅ΡΠΊΠΈ Π½Π΅Ρ Π½ΠΈΠΊΠ°ΠΊΠΈΡ
ΠΏΡΠΎΡΠΈΠ²ΠΎΡΠ΅ΡΠΈΠΉ Ρ ΡΠΎΠ²ΡΠ΅ΠΌΠ΅Π½Π½ΠΎΠΉ ΡΠ΅Π³Π»Π°ΠΌΠ΅Π½ΡΠΈΡΡΡΡΠ΅ΠΉ Π΄ΠΎΠΊΡΠΌΠ΅Π½ΡΠ°ΡΠΈΠ΅ΠΉ Π΄Π»Ρ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ΠΈΡ ΡΡΠΎΠ³ΠΎ ΠΌΠ΅ΡΠΎΠ΄Π° Π² ΠΊΠ°ΡΠ΅ΡΡΠ²Π΅ ΠΎΡΠ½ΠΎΠ²Π½ΠΎΠ³ΠΎ