BIOINFORMATIC SEARCH OF CRISPR/CAS SYSTEM STRUCTURES IN GENOME OF PCT281 PLASMID OF BACILLUS THURINGIENSIS SUBSP. CHINENSIS STRAIN CT-43

Background. CRISPR/Cas systems loci are one of the functionally important patterns in bacterial genome which perform the role of “adaptive immune defense” from foreign nucleic acids. The study of CRISPR/Cas systems structure in genomes of plasmids and phages provide new information about the evolution of this systems in bacterial hosts.Aims. A search of CRISPR/Cas systems structures in pCT281 plasmid from Bacillus thuringiensis subsp. chinensis strain CT-43 using bioinformatic methods.Materials and methods. Search studies using bioinformatics methods were performed with the genome of pCT281 plasmid of B. thuringiensis subsp. chinensis strain CT-43 from the RefSeq database. To search for the CRISPR/Cas system structure MacSyFinder (ver. 1.0.5) and three combined algorithms were used: CRISPRFinder; PILER-CR; CRISPR Recognition Tool (CRT). The consensus repeat sequence was generated in WebLogo 3.Results and discussion. In pCT281 plasmid we detected one locus of CRISPR/Cas system of the type I-C which contains 2 CRISPR-cassettes and 4 cas-genes located between them. The CRISPR-cassette 1 includes 10 spacers from 32 to 35 bp and 11 repeats 32bp in length. 5 spacers (33–35 bp) separated by 6 repeats 32 bp in length were detected in the CRISPR-cassette 2.Conclusions. The bioinformatic methods used in this study enable to conduct a search of CRISPR/Cas systems structures in plasmid genomes. The presence of the CRISPR-Cas locus in pCT281 plasmid confirms a possible transfer of this system from the nucleoid to this plasmid. The detected spacers provide information about phages this bacteria was encountered

Certification and potential future use of the reference standard designed for the test system for determination of the fractional (antigenic) composition of human serum products by immunoelectrophoresis

The article gives an account of certification performed for a new batch of a Reference standard to be used with the test system for determination of the fractional (antigenic) composition of human serum products by immunoelectrophoresis -industry reference standard (IRS) 42-28-77 - in accordance with existing requirements. A candidate reference standard was represented by a batch of normal human serum intended for diagnostic purposes (more than 500 donors) and a batch of immunoelectrophoresis serum against human serum proteins (antiserum against human serum proteins). In accordance with the certification programme, the fractional (antigenic) composition of the reference standard was determined by immunoelectrophoresis using «KliniTest-EF» buffer and 0.05 M borate buffer solution and the modes stipulated in the general monograph 1.8.2.0002.15 Agar gel immunoelectrophoresis. The reaction between the antiserum against human serum proteins and the normal human serum components of the reference standard produced at least 15 precipitation lines. The study demonstrated that the bromphenol blue dye could be used together with «KliniTest-EF» buffer to assess albumin migration, and the pyronin B dye could be used together with «KliniTest-EF» buffer and 0.05 M borate buffer solution to assess immunoglobulin migration. The study confirmed the applicability of the certified reference standard designed for the test system for determination of the fractional (antigenic) composition of human serum products by immunoelectrophoresis, and set the criteria for its use in the context of human serum products identification (species specificity) testing not only by immunoelectrophoresis, but also by agar-gel immunodiffusion

Особенности аттестации и перспективы применения стандартного образца тест-системы для определения фракционного (антигенного) состава препаратов из сыворотки крови человека методом иммуноэлектрофореза

The article gives an account of certification performed for a new batch of a Reference standard to be used with the test system for determination of the fractional (antigenic) composition of human serum products by immunoelectrophoresis -industry reference standard (IRS) 42-28-77 - in accordance with existing requirements. A candidate reference standard was represented by a batch of normal human serum intended for diagnostic purposes (more than 500 donors) and a batch of immunoelectrophoresis serum against human serum proteins (antiserum against human serum proteins). In accordance with the certification programme, the fractional (antigenic) composition of the reference standard was determined by immunoelectrophoresis using «KliniTest-EF» buffer and 0.05 M borate buffer solution and the modes stipulated in the general monograph 1.8.2.0002.15 Agar gel immunoelectrophoresis. The reaction between the antiserum against human serum proteins and the normal human serum components of the reference standard produced at least 15 precipitation lines. The study demonstrated that the bromphenol blue dye could be used together with «KliniTest-EF» buffer to assess albumin migration, and the pyronin B dye could be used together with «KliniTest-EF» buffer and 0.05 M borate buffer solution to assess immunoglobulin migration. The study confirmed the applicability of the certified reference standard designed for the test system for determination of the fractional (antigenic) composition of human serum products by immunoelectrophoresis, and set the criteria for its use in the context of human serum products identification (species specificity) testing not only by immunoelectrophoresis, but also by agar-gel immunodiffusion.Представлены материалы по аттестации новой серии «Стандартного образца тест-системы для определения фракционного (антигенного) состава препаратов из сыворотки крови человека методом иммуноэлектрофореза» ОСО 42-28-77 в соответствии с современными требованиями. В качестве кандидата в стандартный образец использованы серия нормальной сыворотки крови человека для диагностических целей (более 500 доноров) и серия сыворотки для иммуноэлектрофореза против сывороточных белков крови человека (антисыворотка к сывороточным белкам крови человека). В соответствии с Программой аттестации определен фракционный (антигенный) состав кандидата в стандартный образец методом иммуноэлектрофореза с использованием буфера «КлиниТест-ЭФ» и 0,05 М боратного буферного раствора в режимах, регламентированных ОФС. 1.8.2.0002.15 «Иммуноэлектрофорез в агаровом геле». Компонент стандартного образца «антисыворотка к сывороточным белкам крови человека» в реакции с компонентом стандартного образца «нормальная сыворотка крови человека» выявляет не менее 15 линий преципитации. Показана перспективность применения красителя бромфеноловый синий для оценки миграции альбумина с использованием буфера «КлиниТест-ЭФ», а также красителя пиронин В для оценки миграции иммуноглобулина с использованием буферов «КлиниТест-ЭФ» и 0,05 М боратного. Подтверждены критерии возможности использования аттестованного стандартного образца тест-системы для определения фракционного (антигенного) состава препаратов из сыворотки крови человека методом иммуноэлектрофореза, а также установлены критерии его использования для подтверждения их подлинности (видоспецифичности) не только методом иммуноэлектрофореза, но и методом иммунодиффузии в агаре