Selection of IS6110 conserved regions for the detection of Mycobacterium tuberculosis using qPCR and LAMP.

IS6110 insertion sequence is a frequently used target for Mycobacterium tuberculosis detection. However, its sequence variability is studied insufficiently. We aimed to identify the most conservative and variable regions in IS6110 sequences and develop qPCR and LAMP oligonucleotide sets for the conservative regions. Using in-house Python scripts, 3609 M. tuberculosis genome sequences from the NCBI database were aligned; conservative regions were identified to design oligonucleotide sets. IS6110 fragments located within the 31-231 bp region were the most conservative and represented in genomes and were used to design qPCR and LAMP oligonucleotides. The in silico sensitivity of the qPCR oligonucleotides on the whole genome set was 99.1% and 98.4%. For the LAMP primers developed, the sensitivity was 96.9%. For qPCR, the limit of detection with 95% confidence (LoD95%) was four IS6110 copies per reaction, with LoD90% being 200 BCG cells per ml of artificial sputum. For LAMP, LoD95% was 16 copies per reaction, with LoD90% being 400 Mycobacterium bovis Bacille Calmette-Guerin (BCG) cells per ml of artificial sputum. We have demonstrated the IS6110 sequence variability and designed highly sensitive qPCR and LAMP oligonucleotides to detect M. tuberculosis.

Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis.

Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification that is more stable and resistant to DNA amplification inhibitors than conventional PCR. LAMP multiplexing with reverse transcription allows for the single-tube amplification of several RNA fragments, including an internal control sample, which provides the option of controlling all analytical steps. We developed a method of SARS-CoV-2 viral RNA detection based on multiplex reverse-transcription LAMP, with single-tube qualitative analysis of SARS-CoV-2 RNA and MS2 phage used as a control RNA. The multiplexing is based on the differences in characteristic melting peaks generated during the amplification process. The developed technique detects at least 20 copies of SARS-CoV-2 RNA per reaction on a background of 12,000 MS2 RNA copies. The total time of analysis does not exceed 40 min. The method validation, performed on 125 clinical samples of patients' nasal swabs, showed a 97.6% concordance rate with the results of real-time (RT)-PCR assays. The developed multiplexed LAMP can be employed as an alternative to PCR in diagnostic practice to save personnel and equipment time.

Derivatives of Bst-like Gss-polymerase with improved processivity and inhibitor tolerance.

At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNA-binding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants-urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance.

Molecular & genetic characteristics of Mycobacterium tuberculosis strains circulating in the southern part of West Siberia.

BACKGROUND & OBJECTIVES: A complicated epidemiological situation characterized by significantly high tuberculosis (TB) morbidity is observed in West Siberia. This study was aimed to investigate the genetic characteristics of Mycobacterium tuberculosis circulating in the southern part of West Siberia (in the Omsk region). METHODS: From March 2013 to January 2015, 100 isolates of M. tuberculosis were obtained from patients with pulmonary TB living in the Omsk region. Drug susceptibility testing was performed on Lowenstein-Jensen medium (absolute concentration method). Genetic typing of isolates was carried out by variable number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) typing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The genetic types and characteristics of cluster strains were determined using 15 MIRU-VNTR loci. RESULTS: Thirty six VNTR types were found. Twenty six (26.0%) isolates had a unique profile, and the remaining 74 were grouped in 10 clusters containing from 2 to 23 isolates. The Beijing genotype was found in 72 isolates, 61 (85.0%) of which were part of five clusters that included two large clusters containing 23 isolates. Other genetic families, such as Latin-American Mediterranean (LAM, 11.0%), S family (2.0%) and Haarlem (4.0%), were also detected. The genetic family of 11 isolates could not be determined. Six different VNTR profiles were found in these non-classified isolates. Only 16 per cent of isolates were sensitive to anti-TB drugs. The katG315 (94.8%) and rpoB531 (92.2%) mutations were identified in 77 multidrug-resistant M. tuberculosis isolates. INTERPRETATION & CONCLUSIONS: This study showed that the M. tuberculosis population in the Omsk region was heterogeneous. The Beijing genotype predominated and was actively spreading. The findings obtained point to the need for the implementation of more effective preventive measures to stop the spread of drug-resistant M. tuberculosis strains.

Use of the VH6-1 gene segment to code for anti-interleukin-18 autoantibodies in multiple sclerosis.

We investigated whether levels and repertoires of anti-interleukin-18 (IL-18) autoantibodies (auto-Abs) differ in multiple sclerosis (MS) patients and healthy donors (HDs). IL-18 concentration in MS patients' sera was higher than in HD, but the level of anti-IL-18 auto-Abs was lower in MS patients. Correlation patterns of IL-18/anti-IL-18 auto-Abs system differed in HD and MS patients, so we have compared segment composition of the anti-IL-18 single-chain variable fragments (scFvs) selected from MS and naïve phage display libraries. Considerable differences between anti-IL-18 auto-Abs of these libraries were found. MS panel contained auto-Abs displaying both signs of "fetal" and somatically hypermutated repertoires. Naïve panel mainly contained the naïve antibodies. These variations from the norm are possible results of abnormal regulation of the repertoire perhaps determined by remodeling of the molecular mechanisms involved in the V(D)J recombination and/or abnormal selection by antigen in MS pathogenesis.

Association of the MTHFR 1298A>C (rs1801131) polymorphism with speed and strength sports in Russian and Polish athletes.

It has been suggested that DNA hypomethylation because of poorer effectiveness of the 5,10-methylenetetrahydrofolate reductase (MTHFR) enzyme induces muscular growth. We hypothesised that the common, functional 1298A>C polymorphism in the MTHFR gene is associated with athletic status. To test this hypothesis, we investigated the distribution of the 1298A>C variant in Polish (n = 302) and Russian (n = 842) athletes divided into four groups: endurance, strength-endurance, sprint-strength and strength-endurance, as well as in 1540 control participants. We found different genotypes (the AC heterozygote advantage) and allele distributions among sprint-strength athletes and strength athletes than the groups of sedentary controls for each nationality. In the combined study, the allelic frequencies for the 1298C variant were 35.6% in sprint-strength athletes (OR 1.18 [1.02-1.36], P = 0.024 vs. controls) and 38.6% in strength athletes (OR 1.34 [1.10-1.64], P = 0.003 vs. controls). The results of the initial and repetition studies as well as the combined analysis suggest that the functional 1298A>C polymorphism in the MTHFR gene is associated with athletic status. The presence of the C allele seems to be beneficial in sprint-strength and strength athletes. It needs to be established whether and to what extent this effect is mediated by alteration in DNA methylation status.

Detecting Microsatellite Instability in Endometrial, Colon, and Stomach Cancers Using Targeted NGS.

PURPOSE: To develop a method for testing the MSI based on targeted NGS. METHODS: Based on the results of previous studies, 81 microsatellite loci with high variability in MSI-H tumors were selected, and a method for calculating the MSI score was developed. Using the MSI score, we defined the MSI status in endometral (162), colon (153), and stomach (190) cancers. Accuracy of the MSI scores was evaluated by comparison with MMR immunohistochemistry for 137 endometrium (63 dMMR and 74 pMMR), 76 colon (29 dMMR and 47 pMMR), and 81 stomach (8 dMMR and 73 pMMR) cancers. RESULTS: Classification of MSS and MSI-H tumors was performed with AUC (0.99), sensitivity (92%), and specificity (98%) for all tumors without division into types. The accuracy of MSI testing in endometrial cancer was lower than for stomach and colon cancer (0.98, 87%, and 100%, respectively). The use of 27 loci only, the most informative for endometrial cancer, increased the overall accuracy (1.00, 99%, and 99%). Comparison of MSI score values in 505 tumors showed that MSI score is significantly higher in colon (p < 10-5) and stomach (p = 0.008) cancer compared with endometrial cancer. CONCLUSION: The MSI score accurately determines MSI status for endometrial, colon, and stomach cancers and can be used to quantify the degree of MSI.

Appearances are deceptive: Three RNA viruses co-infected with the nucleopolyhedrovirus in host Lymantria dispar.

The virus infection, which visually looks like typical monoinfection, in fact may hide a great complex of different species. Without detailed analysis, we may miss the important interaction between pathogens, including new species. In the current study, we found the new species inside the mix of cubic and polyhedral occlusion bodies (OBs) isolated from the gypsy moth, Lymantria dispar L. (Ld). Transmission electron microscopy (TEM) revealed that into the one cadaver were OBs which belonged to baculovirus and cypoviruses. The baculovirus produced polyhedral OBs, while cypoviruses produced polyhedral and cubic OBs. Genomic analysis detected the multiple Ld nucleopolyhedroviruses, and cypoviruses were Hubei lepidoptera virus 3 and Dendrolimus punctatus cypovirus 1. This represents the first isolation of the Hubei lepidoptera virus 3 from the gypsy moth, proposed as "Lymantria dispar cypovirus 3". The RNAseq analysis also revealed the presence of Lymantria dispar iflavirus 1. The insecticidal activity of the mixed infection was comparable to that of typical baculovirus monoinfection. Thus, we demonstrate that i) the shape of OBs identified by light microscopy cannot be a robust indicator of viral species infecting the host; ii) only specific analysis may reveal the true composition of viral infection.

The attachment of a DNA-binding Sso7d-like protein improves processivity and resistance to inhibitors of M-MuLV reverse transcriptase.

Reverse transcriptases (RTs) are a standard tool in both fundamental studies and diagnostics. RTs should possess elevated temperature optimum, high thermal stability, processivity and tolerance to contaminants. Here, we constructed a set of chimeric RTs, based on the combination of the Moloney murine leukaemia virus (M-MuLV) RT and either of two DNA-binding domains: the DNA-binding domain of the DNA ligase from Pyrococcus abyssi or the DNA-binding Sto7d protein from Sulfolobus tokodaii. The processivity and efficiency of cDNA synthesis of the chimeric RT with Sto7d at the C-end are increased several fold. The attachment of Sto7d enhances the tolerance of M-MuLV RT to the most common amplification inhibitors: NaCl, urea, guanidinium chloride, formamide, components of human whole blood and human blood plasma. Thus, fusing M-MuLV RT with an additional domain results in more robust and efficient RTs.

Comparison of fluorescent intercalating dyes for quantitative loop-mediated isothermal amplification (qLAMP).

Real-time or quantitative loop-mediated isothermal amplification (qLAMP) is a promising technique for the accurate detection of pathogens in organisms and the environment. Here we present a comparative study of the performance of six fluorescent intercalating dyes-SYTO-9, SYTO-13, SYTO-82, SYBR Green I, SYBR Gold, EvaGreen-in three different qLAMP model systems. SYTO-9 and SYTO-82, which had the best results, were used for additional enzyme and template titration studies. SYTO-82 demonstrated the best combination of time-to-threshold (Tt) and signal-to-noise ratio (SNR).

Large Fragment of DNA Polymerase I from Geobacillus sp. 777: Cloning and Comparison with DNA Polymerases I in Practical Applications.

A truncated gene of DNA polymerase I from the thermophilic bacteria Geobacillus sp. 777 encoding a large fragment of enzyme (LF Gss pol) was cloned and sequenced. The resulting sequence is 1776-bp long and encodes a 592 aa protein with a predicted molecular mass of 69.8 kDa. Enzyme was overexpressed in E. coli, purified by metal-chelate chromatography, and biochemically characterized. The specific activity of LF Gss pol is 104,000 U/mg (one unit of enzyme was defined as the amount of enzyme that incorporated 10 nmol of dNTP into acid insoluble material in 30 min at 65 °C). The properties of LF Gss pol were compared to commercially available large fragments of DNA polymerase I from G. stearothermophilus (LF Bst pol) and Bacillus smithii (LF Bsm pol). Studied enzymes showed maximum activity at similar pH and concentrations of monovalent/divalent ions, whereas LF Gss pol and LF Bst pol were more thermostable than LF Bsm pol. LF Gss pol is more resistant to enzyme inhibitors (SYBR Green I, heparin, ethanol, urea, blood plasma) in comparison with LF Bst pol and LF Bsm pol. LF Gss pol is also suitable for loop-mediated isothermal amplification and whole genome amplification of human genomic DNA.