DNA Amplification Methods Research Articles

Electrochemical Detection of Genomic DNA Utilizing Recombinase Polymerase Amplification and Stem-Loop Probe.

Nucleic acid tests integrated into digital point-of-care (POC) diagnostic systems have great potential for the future of health care. However, current methods of DNA amplification and detection require bulky and expensive equipment, many steps, and long process times, which complicate their integration into POC devices. We have combined an isothermal DNA amplification method, recombinase polymerase amplification, with an electrochemical stem-loop (S-L) probe DNA detection technique. By combining these methods, we have created a system that is able to specifically amplify and detect as few as 10 copies/μL Staphylococcus epidermidis DNA with a total time to result of 70−75 min.

Comparative Assessment of DNA Targets and Amplification Methods for Leishmania (Viannia) Detection in Human Samples.

.Multiple polymerase chain reaction (PCR)-based approaches have been developed for Leishmania detection in clinical and laboratory samples, and this diversity limits inter-study comparisons, meta-analyses, and generalization of findings. Towards harmonization of a molecular tool for detection of Leishmania (Viannia) for research purposes, we evaluated the concordance of 18SrDNA quantitative polymerase chain reaction (qPCR) and minicircle kinetoplastid DNA (mkDNA) PCR followed by Southern blot (PCR-SB) in in vitro infection systems and in lesion and mucosal swab samples from Colombian patients with cutaneous leishmaniasis caused by L. (Viannia). The lower limit of parasite detection of 18SrDNA qPCR and mkDNA PCR-SB was 10−1 promastigotes and one intracellular amastigote per reaction. From cutaneous lesions (n = 63), an almost perfect concordance was found between the methods (κ = 0.92, 95% CI: 0.82–1.00). Despite equal limits of detection, mkDNA PCR-SB was more efficient for parasite detection in mucosal samples than 18SrDNA qPCR or 18SrDNA digital droplet PCR. The high concordance, sensitivity, scaling potential, and feasibility of implementation of the 18SrDNA qPCR, support its selection as the L. (Viannia) in research laboratories, as a first step towards harmonization of research protocols in the region.

Loop-mediated isothermal amplification (LAMP) assays for Anopheles funestus group and Anopheles gambiae complex species.

Species of the genus Anopheles vary with regard to their vector capacity for Plasmodium spp., the causative agent of malaria, and their accurate identification is often required. Loop-mediated isothermal amplification (LAMP) is a rapid, simple and low-cost method for specific DNA amplification. Primers for LAMP assays specific for the Anopheles funestus group and Anopheles gambiae complex species as well as for the species Anopheles arabiensis, An. funestus, An. gambiae s.s/Anopheles coluzzii (major vectors) and Anopheles rivulorum (minor vector) were designed targeting specific genome or rDNA internal transcribed spacer regions. Reaction conditions (buffer composition, primer concentrations, incubation time) were evaluated and the specificities of the assays confirmed with DNA from non-target Anopheles species. DNA release from the mosquitoes is achieved simply by heating them for 5 min in water. An aliquot of the DNA solutions is transferred to the reaction tube using disposable inoculation loops. The outcome of the LAMP amplifications after 1 h incubation at 65 °C can easily be visualized by a colour change visible to the naked eye. The assays are operable under field conditions requiring only basic equipment (portable heat block programmable at 65 and 80 °C, cooler for master mixes).

New pathogens causing CNS infections

Many emerging infections, mainly viruses, are contributing to the reshaping of the global landscape of central nervous system (CNS) infections. Several new organisms causing CNS infection have been discovered over the last decade, and new neurological syndromes have been described for many other known pathogens. Advances in diagnostic techniques such as modern DNA amplification methods have led to the detection of some of the newly identified pathogens. The global attention generated by disease epidemics has helped in the improved understanding of the neurological complications associated with some known pathogens, such as Zika virus, Ebola virus and Enterovirus D68. A better understanding of the evolution of the new pathogens, their hostvector- environment dynamics, and their disease causation is clearly needed for us to be better prepared to meet these emerging threats.

Association of HLA-G 3'UTR 14-bp Ins/Del polymorphism with breast cancer among South Indian women

AimHuman leucocyte antigen-G (HLA-G) and tumour necrosis factor-alpha (TNF-α) are potent immune mediators implicated in the pathogenesis of breast cancer. The polymorphisms in the 3' untranslated region (3'UTR) of HLA-G...

Applying a Linear Amplification Strategy to Recombinase Polymerase Amplification for Uniform DNA Library Amplification.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification method with broad applications as a point-of-care test and in molecular biology techniques. Currently, most of the applications are focused on target-specific amplification. Because RPA has the advantage of amplifying DNA under isothermal conditions, we utilized RPA as a DNA library amplification tool. In this study, we used a sheared genomic DNA library and an oligonucleotide (oligo) library for the comparison of polymerase chain reaction and RPA. For the sheared DNA library, we observed biased amplification after RPA was conducted. Thus, to amplify the size-variable DNA library uniformly, we introduced a linear amplification strategy with RPA and successfully improved the uniformity. On the other hand, using the same-sized oligo library, we confirmed that RPA amplified this library uniformly without modification of the protocol. These results demonstrate that RPA can be applied not only to amplify a specific target as previously demonstrated but also to amplify a complex DNA library composed of a large number of different DNA molecules.

An enhanced DNA amplification method to detect the species origins of the rootless hair shafts

Hairs are commonly encountered at crime scenes. Determining the biological origins of hairs is important for case investigations. However, the DNA extraction of rootless hair shafts are quite difficult. In this study, an enhanced DNA amplification methodology is revealed, in combination with a self-developed system of 5 × PCR Reaction Master Mix IV, to directly amplify the mtDNA 12S ribosomal RNA (12S rRNA) gene from rootless hair shafts collected from 12 common species (human, chicken, duck, cattle, sheep, dog, mouse, pig, horse, cat, rabbit and pigeon) without DNA extraction. The amplicons were subsequently sequenced on Pyromark Q48 (QIAGEN, Hilden, Germany). The results illustrated that the direct amplification system could efficiently amplify the targeted mtDNA genes and pyrosequencing technology could further readily differentiate the sample origin of the rootless hair shafts. Data analysis of the 12S rRNA was equipped to an automatic analysis module. This study provides a new solution for species identification of rootless hair shafts.

Detection of Candida Albicans in some enteropathy patients using PCR technique

Laboratory identification based on DNA amplification methods by used PCR may provide an alternative and more sensitive method than traditional culture for the early detection of Candida albicans. This study aims to the identification and determines the prevalence of C. albicans by use PCR method. A total of 167 samples (40 controls and 127 patients) were taken .By the use of culture techniques for isolation and identification C. albicans on Sabouraud dextrose agar are characterized by cream to white and smooth at 30C◦ these characteristics. C. albicans have been isolated from patients' samples and these included the C.albicans was detected in 17 control subjects (42.5%), and in 51 patients (40.16%). The results of this estimation revealed that the amplified DNA (PCR product) was 459bp for C.albicans.

Assessing the comparability of different DNA extraction and amplification methods in gut microbial community profiling.

Automated, high-throughput technologies are becoming increasingly common in microbiome studies to decrease costs and increase efficiency. However, in microbiome studies, small differences in methodology – including storage conditions, wet lab methods, sequencing platforms and data analysis – can influence the reproducibility and comparability of data across studies. There has been limited testing of the effects of high-throughput methods, including microfluidic PCR technologies. In this paper, we compare two extraction methods (the QIAamp DNA Stool Mini Kit and the MoBio PowerSoil DNA Isolation kit), two taq polymerase enzymes (MyTaq HS Red Mix and Accustart II PCR ToughMix), two primer sets (V3–V4 and V4–V5) and two amplification methods (a common two-step PCR protocol and amplicon library preparation on the Fluidigm Access Array system that allows automated multiplexing of primers). Gut microbial community profiles were significantly affected by all variables. While there were no significant differences in alpha diversity measured between the two extraction methods, there was an effect of extraction method on community composition measured by unweighted UniFrac distances. Both amplification method and primers had a significant effect on both alpha diversity and community composition. The relative abundance of Actinobacteria was significantly lower when using the MoBio kit or Fluidigm amplification method, and the relative abundance of Firmicutes was lower when using the Qiagen kit. Microbial community profiles based on Fluidigm-generated amplicon libraries were not comparable to those generated with more commonly used methods. Researchers should carefully consider the limitations and biases that different extraction and amplification methods can introduce into their results. Additionally, more thorough benchmarking of automated and multiplexing methods is necessary to determine the magnitude of the potential trade-off between the quality and the quantity of data.

Abstract 3519: High-throughput unbiased microwell-based single-cell CNV detection reveals tumor clonal subtypes in hepatocellular carcinoma

Abstract Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing DNA amplification and library preparation methods are expensive and introduce coverage bias during sample pre-amplification. Based on a massively parallel 5184-microwell chip, we developed a high-throughput unbiased single-cell DNA library preparation approach up to 1800 cells for copy number variation (CNV) detection, which provides a comprehensive, scalable solution for revealing genome heterogeneity. Compared with existing methods, it is more efficient and cost-effective to construct a barcoded-single-cell library without pre-amplification in 2 days. Besides, it has significantly lower Median Absolute Pairwise Difference (MAPD:0.17±0.004) values which are used to evaluate the amplification biases and noises, than using MDA (multiple displacement amplification) method (MAPD: 0.79±0.23) or MALBAC (multiple annealing and looping-based amplification cycles) approach (MAPD : 0.24±0.002). We further successfully applied this approach to obtain single-cell CNV information of more than 500 cells from a tumor sample of hepatocellular carcinoma in one run. Using the unsupervised clustering, we identified at least two clonal subpopulations with a multitude of non-integer alterations across the genome. Overall, this is an unbiased, effective, high-throughput and low cost approach to figure out tumor heterogeneity and understand clonal evolution in tumor at the single-cell level. Citation Format: Liang Wu, Yuzhou Wang, Miaomiao Jiang, Shiping Liu. High-throughput unbiased microwell-based single-cell CNV detection reveals tumor clonal subtypes in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3519.

Ten-minute direct detection of Zika virus in serum samples by RT-LAMP

Zika virus (ZIKV) is a current threat to global health. In most of cases, ZIKV infection has no symptoms; however in some cases, ZIKV can cause paralysis (Guillain-Barré syndrome), and in pregnant women, it can cause birth defects in infants. Rapid and accurate diagnosis can help improve disease control as well as being vital to prenatal care for women living in endemic areas. Molecular diagnostics based on isothermal amplification techniques are an excellent alternative to conventional methods of DNA amplification, such as PCR. Here, we develop and optimized a rapid and sensitive method for direct detection of ZIKV in Serum samples based on RT-LAMP and visual detection. The reaction was thermally controlled with a thermoblock for 10 min at 72 °C. The results show that the use of the Bst 3.0 enzyme and an adequate optimization can further reduce the time needed for the RT-LAMP reaction to detect ZIKV. Our results demonstrate that it is possible to detect ZIKV through RT-LAMP directly from a Serum sample, without prior RNA extraction. As little as 10-3 copies of RNA in a 10 μL reaction (20 zepto-molar) was detected by RT-LAMP from a panel of 51 Serum samples (16 samples from pregnant women and 35 samples from newborns infected with ZIKV during pregnancy). The RT-LAMP has proven to be a valuable tool for molecular diagnosis of Zika, presenting a great potential for point-of-care applications, especially in developing countries.

Identification and Discrimination of Salmonella Enteritidis, S. Pullorum, S. Gallinarum and S. Dublin Using Salmonella Specific Genomic Regions Amplification Assay

Background: DNA amplification method has been developed for identifying and discriminating Salmonella serovars, using specific primers at the genus and serovar levels and to identify the S. Enteritidis, S. Dublin, S. Gallinarum and S. Pullorum. Objectives: This study was conducted for molecular identification and discrimination among some important Salmonella serovars. Methods: Fifty isolates of Salmonella were assayed. The PCR assay was designed to amplify DNA fragments from six Salmonella genes, invA (284 bp), tcpS (882 bp), lygD (339 bp), flhB (155 bp), SlgC (252 bp), and speC (174 bp). Results: The results showed invA and tcpS genes presence in all four Salmonella serovars, whereas the lygD gene only exists in S. Enteritidis and is not found in S. Dublin, S. Gallinarum and S. Pullorum. The flhB gene is only present in S. Enteritidis and S. Dublin whereas it does not exist in S. Gallinarum and S. Pullorum. The SlgC gene exists in both S. Gallinarum and S. Pullorum, the SpeC gene is specifically present in S. Gallinarum, whereas SlgC and SpeC genes are not found in S. Enteritidis and S. Dublin. Salmonella Dublin serovar amplification assay successfully identified three selected serovar specific genomics regions (SSGRs) and hut gene. The results identify hut gene (495 bp), DSR1 (Dublin-specific genomics region1) (105 bp), DSR2 (Dublin-specific genomics region2) (203 bp), and DSR3 (Dublin-specific genomics region3) (296 bp). Conclusions: Amplification techniques on Salmonella serovars specific genomics regions are able to identify and discriminate clinically significant Salmonella serovars, and therefore, have the possibility to be used as a useful and rapid screening assay and support conventional biochemical and serological examinations

Microfluidics-Based Enrichment and Whole-Genome Amplification Enable Strain-Level Resolution for Airway Metagenomics.

Dysbiosis of airway microbiomes has been found in various respiratory diseases, but its molecular details in terms of taxonomic profile, metabolic characteristics, defensive function, and inhabit adaption are far from clear. Shotgun metagenome sequencing provides detailed information for microbes, whereas its application is rather limited in airways due to host DNA contaminants that overwhelm a minute amount of microbial content. Here, we describe a microfluidics-based enrichment device and an emulsion-based whole-genome amplification procedure (MEEA) for the preparation of DNA from sputa for shotgun sequencing in a metagenomics study. The two protocols coupled in MEEA are first separately assayed with mock samples and are both promising in efficiency and bias. The efficiency and consistency of MEEA are further evaluated in six clinical sputum samples against direct sequencing without enrichment, and MEEA enables 2 to 14 times enrichment for microbial reads, which take 14.68% to 33.52% of total reads. The dominant pathogens detected in MEEA are in excellent agreement with those from clinical etiological tests. Meanwhile, MEEA presents much more microbiome complexity and genome information at a strain level than direct sequencing, exhibiting high sensitivity for identifying prophages and DNA viruses. MEEA provides better microbiome profiling than direct sequencing without a preference for specific microorganisms. The more detailed functional and taxonomic characterization of their species constituents, including both bacterium and virus, facilitates metagenomics studies on the pathogenesis of respiratory microbiomes.IMPORTANCE The airway microbial community, which takes important pathogenic roles for respiratory diseases, is far from clear in terms of taxonomy and gene functions. One of the critical reasons is the heavy contamination of host cell/DNA in airway samples, which hinders the subsequent sequencing of the whole genomic contents of the microbial community-the metagenome. Here, we describe a protocol for airway sample preparation which couples a microbe enrichment microfluidic device and a DNA amplification method performed in numerous droplets. When evaluated with mock and clinical sputum samples, the microfluidics-based enrichment device and emulsion-based whole-genome amplification (MEEA) procedure efficiently removes host cells, amplifies the microbial genome, and shows no obvious bias among microbes. The efficiency of MEEA makes it a promising method in research of respiratory microbial communities and their roles in diseases.

The possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods

Causative agents of melioidosis and glanders are among the most dangerous bacterial pathogens for human. Moreover, Burkholderia pseudomallei and Burkholderia mallei are considered to be potential bioterrorism agents. In connection with this, timely diagnostics of such bacteria is of high importance. In our study, we made an attempt to develop an approach for detecting pathogenic Burkholderia spp. by combining species-specific amplification and strain-specific dot blotting assay with monoclonal antibodies. The following pathogenic Burkholderia strains were used in experiments: B. mallei (C-4, C-5, t-12, B-120, P-1, Мuksuwar-11, Z-12,Zagreb, Ivanovich, 5534), and B. pseudomallei (100, 102, 115, 116, 132, 135, 301, 51274, 60913, 61503). Real-Time PCR (RT-PCR) and dot blotting with monoclonal antibodies against surface Burkholderia epitopes were used to detect such pathogens. RT-PCR was carried out by using primers designed to recognize DNA fragments in B. mallei IS407A-fliP and the gene Orf12 from B. pseudomallei. For this, DNA was isolated from bacterial cells suspended at 1 × 104 microbial cells/ml. accumulation of the end reaction products was visualized by staining with dye SYBR Green I. Specificity of amplification reaction was determined by measuring melting temperature (Tm) for end products followed by running gel electrophoresis. It was demonstrated that all ten strains of either B. mallei or B. pseudomallei examined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. It was demonstrated that all ten strains of either B. mallei or B. pseudomallei examined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. Importantly, no signals specific to heterologous microbial DNA (isolated from bacterial cell suspension at concentration of 1 × 107 microbial cells/ml) were detected by using RT-PCR. Thus, RT-PCR provides an opportunity for assessing an inter-species diversity among pathogenic Burkholderia species. A genus-specificity was observed by using monoclonal antibodies 3D3 which bind to both Burkholderia strains, whereas antibodies 2D11 exhibited no selective binding to strain Р1 B. mallei and strain 100 B. pseudomallei, thereby displaying a strain-specific interaction. Thus, it allowed to conclude that combining a species-specific DNA amplification particularly RT-PCR together with immune-based assay such as dot blotting by using a panel of monoclonal antibodies seems to be a promising approach for assessing intra-species diversity among pathogenic Burkholderia.

Ancient DNA (aDNA) extraction and amplification from 3500-year-old charred economic crop seeds from Kaymakçı in Western Turkey: comparative sequence analysis using the 26S rDNA gene

Ancient DNA (aDNA) from 3500–4000 years old seeds of Triticum aestivum L. or Triticum durum Dest., Vicia ervillia (L) Willd., Cicer arietinum L. and Vitis vinifera L. excavated from the archaeological site of Kaymakci was successfully extracted using various isolation methods. The genomic DNA of each species was amplified with respect to the 26S ribosomal DNA (rDNA) gene further using the aDNA of the seeds. The reasons for successful DNA extraction and amplification are likely due to (1) preservation of certain ancient seed specimens in good conditions and (2) use of improved DNA extraction and amplification methods. The results indicate that all seeds were identified correctly by the DNA sequence data from the 26S rDNA gene. Specifically, a morphologically unidentified wheat seed from Kaymakci was characterized by DNA sequence data as bread wheat (Triticum aestivum). Comparative sequence analysis revealed that specific base positions in the ancient 26S rDNA gene were either lost or substituted with different DNA bases in contemporary seeds, most likely due to continued domestication and breeding activities. Attaining high amounts and a good quality of amplified genomic DNA from ancient seeds will further allow the investigation of the extent of genetic change between ancient seeds and their contemporary species in genetic diversity studies.

A mathematical model for a biphasic DNA amplification reaction.

Isothermal DNA amplification reactions are a prevalent tool with many applications, ranging from analyte detection to DNA circuits. Exponential amplification reaction (EXPAR) is a popular isothermal DNA amplification method that exponentially amplifies short DNA oligonucleotides. A recent modification of this technique using an energetically stable looped template with palindromic binding regions demonstrated unexpected biphasic amplification and much higher DNA yield than EXPAR. This ultrasensitive DNA amplification reaction (UDAR) shows high-gain, switch-like DNA output from low concentrations of DNA input. Here we present the first mathematical model of UDAR based on four reaction mechanisms and show the model can reproduce the experimentally observed biphasic behaviour. Furthermore, we show that three of these mechanisms are necessary to reproduce biphasic experimental results. The reaction mechanisms are (i) positively cooperative multistep binding spurred by two trigger binding sites on the template; (ii) gradual template deactivation; (iii) recycling of deactivated templates into active templates; and (iv) polymerase sequestration. These mechanisms can potentially illuminate the behaviour of EXPAR as well as other nucleic acid amplification reactions.

Non-invasive surveillance of Plasmodium infection by real-time PCR analysis of ethanol preserved faeces from Ugandan school children with intestinal schistosomiasis

BackgroundAs part of ongoing co-surveillance of intestinal schistosomiasis and malaria in Ugandan school children, a non-invasive detection method for amplification of Plasmodium DNA using real-time (rt)PCR analysis of ethanol preserved faeces (EPF) was assessed. For diagnostic tabulations, results were compared to rtPCR analysis of dried blood spots (DBS) and field-based point-of-care (POC) rapid diagnostic tests (RDTs).MethodsA total of 247 school children from 5 primary schools along the shoreline of Lake Albert were examined with matched EPF and DBS obtained. Mean prevalence and prevalence by school was calculated by detection of Plasmodium DNA by rtPCR using a 18S rDNA Taqman® probe. Diagnostic sensitivity, specificity, positive and negative predictive values were tabulated and compared against RDTs.ResultsBy rtPCR of EPF and DBS, 158 (63.9%; 95% CI 57.8–69.7) and 198 (80.1%, 95% CI 74.7–84.6) children were positive for Plasmodium spp. By RDT, 138 (55.8%; 95% CI 49.6–61.9) and 45 (18.2%; 95% CI 13.9–23.5) children were positive for Plasmodium falciparum, and with non-P. falciparum co-infections, respectively. Using RDT results as a convenient field-based reference, the sensitivity of rtPCR of EPF and DBS was 73.1% (95% CI 65.2–79.8) and 94.2% (95% CI 88.9–97.0) while specificity was 47.7% (95% CI 38.5–57.0) and 37.6% (95% CI 29.0–46.9), respectively. With one exception, school prevalence estimated by analysis of EPF was higher than that by RDT. Positive and negative predictive values were compared and discussed.ConclusionsIn this high transmission setting, EPF sampling with rtPCR analysis has satisfactory diagnostic performance in estimation of mean prevalence and prevalence by school upon direct comparison with POC-RDTs. Although analysis of EPF was judged inferior to that of DBS, it permits an alternative non-invasive sampling regime that could be implemented alongside general monitoring and surveillance for other faecal parasites. EPF analysis may also have future value in passive surveillance of low transmission settings.

Detection of rifampicin resistance of Mycobacterium tuberculosis using multiplex allele specific polymerase chain reaction (MAS-PCR) in Pakistan

Drug resistance in tuberculosis (TB) is a major public health challenge in developing countries such as Pakistan. Multiplex allele specific polymerase chain reaction (MAS-PCR) is a DNA amplification method that could contribute to rapid detection and control of drug resistant tuberculosis (DR-TB) in Pakistan. The purpose of this study was to test the utility of MAS-PCR to detect resistance in Pakistan. Drug susceptibility testing (DST) was used to identify rifampicin resistant and susceptible clinical isolates from TB cases in Pakistan. MAS-PCR was used to detect the most frequent mutations in the gene rpoB among 213 resistant and 37 susceptible isolates. Among 213 clinical isolates, MAS-PCR identified mutation D435Y (Asp435Tyr) in 24 (11.3%) cases, H445Y (His445Tyr) in 14 (6.6%), S450L (Ser450Leu) in 124 (58.2%) and S450W (Ser450Trp) in 18 (8.4%) cases. MAS-PCR did not detect known mutations in 33 (15.5%) cases. Among 12 cases, a novel mutation at codon 434 (Met434Ile) and a common variant at codon 435 (Asp435Tyr) was detected in rpoB gene which is indicative of double mutation. In 4 isolates, a novel mutation at codon 432 (Gln432Pro) was identified. In an additional 4 isolates, mutations Met434Val and His445Asn were identified. Moreover, a mutation in rpoB (Leu452Pro) was found in 5 isolates. DNA sequencing confirmed the absence of mutations in rpoB in the 8 remaining isolates. MAS-PCR had 88.3% sensitivity and 100% specificity using DST as the reference, which suggested that this method could be implemented as an initial marker for screening of multi-drug resistant tuberculosis (MDR-TB) in Pakistan.

Detection of ser450leu mutation in rpoB gene of mycobacterium tuberculosis by allele-specific loop-mediated isothermal dna amplification method

Для выявления генетических мутаций широко используют достаточно трудоемкий и дорогостоящий метод полимеразной цепной реакции (ПЦР). Целью работы было оценить возможность применения двух схем метода аллель-специфичной изотермической петлевой амплификации (loop-mediated isothermal amplification, LAMP) для выявления мутации TCG/TTG (S450L) в гене rpoB Mycobacterium tuberculosis. Использовали 48 клинических изолятов M. tuberculosis и 11 образцов мокроты, выбранных случайным образом и полученных в микробиологической лаборатории г. Новосибирска от пациентов с впервые выявленным заболеванием. Показано, что применение схемы анализа с использованием аллель-специфичного праймера FIP по сравнению с F3 имеет лучшую разрешающую способность: разница между временем амплификации мутации и аллеля дикого типа составила 22 ± 2,4 против 13 ± 4,1 мин (p = 0,0011). При использовании 100 геном-эквивалентов ДНК истинно положительный сигнал (амплификация гена rpoB с мутацией при использовании соответствующего аллель-специфического праймера) детектировался после 29,4 ± 3,4 мин. Положительный сигнал визуализировался после добавления в реакцию SYBR Green I, как при освещении дневным светом, так и при использовании трансиллюминатора с УФ-излучением. С помощью разработанного нами метода была проанализирована выборка ДНК 20 RIFR изолятов M. tuberculosis, несущих мутацию Ser450Leu в гене rpoB, 10 RIFR изолятов, несущих другие мутации в гене rpoB, а также 18 RIFs изолятов без мутаций; наличие мутаций в образцах было определено с помощью классического секвенирования по Сенгеру. Чувствительность и специфичность LAMP для выявления мутации Ser450Leu в гене rpoB составили 100%. Данный подход позволяет использовать в качестве ДНК грубые лизаты микобактерий, что сокращает тотальное время анализа до 1,5 ч.

Detection of Ser450Leu mutation in rpoB gene of Mycobacterium tuberculosis by allele-specific loop-mediated isothermal DNA amplification method

To identify genetic mutations a rather time-consuming and expensive method of polymerase chain reaction (PCR) is widely used. The aim of the present work was to evaluate the possibility of using the two schemes of the method of allele-specific isothermal loop amplification (LAMP) to detect the TCG/TTG (S450L) mutation in the rpoB gene of Mycobacterium tuberculosis. 48 clinical isolates of M. tuberculosis and 11 samples of sputum were used, randomized and obtained in the microbiological laboratory of the city of Novosibirsk from incident patients. It is shown that the use of an analysis scheme using the allele-specific primer FIP compared to F3 has the best resolution: the difference between the amplification time of the mutation and the wild type allele was 22 ± 2,4 versus 13 ± 4,1 minutes (p = 0,0011). When using 100 DNA genomic equivalents a true positive signal (amplification of the rpoB gene with a mutation using the corresponding allele-specific primer) was detected after 29,4 ± 3,4 minutes. A positive signal was visualized after adding SYBR Green I to the reaction, both when illuminated with daylight and when using a UV transilluminator. Using the developed method the DNA sample of 20 RIFR isolates from M. tuberculosis was analyzed containing the Ser450Leu mutation in the rpoB gene, 10 RIFR isolates containing other mutations in the rpoB gene and 18 RIFs isolates without any mutations; the presence of mutations in the samples was determined using classical Sanger sequencing. The sensitivity and specificity of LAMP for detecting a Ser450Leu mutation in the rpoB gene was 100%. This approach allows the use of crude lysates of mycobacteria as DNA, which reduces the total analysis time to 1,5 hour.