Optimizing and accelerating the assignation of lineages in Mycobacterium tuberculosis using novel alternative single-tube assays
María CarcelénEstefanía AbascalMarta HerránzSheila SantantónRoberto Zenteno‐CuevasMaría José SerranoEmilio BouzaLaura Pérez‐LagoDarı́o Garcı́a de Viedma
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Abstract:
The assignation of lineages in Mycobacterium tuberculosis (MTB) provides valuable information for evolutionary and phylogeographic studies and makes for more accurate knowledge of the distribution of this pathogen worldwide. Differences in virulence have also been found for certain lineages. MTB isolates were initially assigned to lineages based on data obtained from genotyping techniques, such as spoligotyping or MIRU-VNTR analysis, some of which are more suitable for molecular epidemiology studies. However, since these methods are subject to a certain degree of homoplasy, other criteria have been chosen to assign lineages. These are based on targeting robust and specific SNPs for each lineage. Here, we propose two newly designed multiplex targeting methods-both of which are single-tube tests-to optimize the assignation of the six main lineages in MTB. The first method is based on ASO-PCR and offers an inexpensive and easy-to-implement assay for laboratories with limited resources. The other, which is based on SNaPshot, enables more refined standardized assignation of lineages for laboratories with better resources. Both methods performed well when assigning lineages from cultured isolates from a control panel, a test panel, and a problem panel from an unrelated population, Mexico, which included isolates in which standard genotyping was not able to classify lineages. Both tests were also able to assign lineages from stored isolates, without the need for subculture or purification of DNA, and even directly from clinical specimens with a medium-high bacilli burden. Our assays could broaden the contexts where information on lineages can be acquired, thus enabling us to quickly update data from retrospective collections and to merge data with those obtained at the time of diagnosis of a new TB case.Keywords:
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Objective To develop a method for genotyping of the human platelet antigen(HPA)-2,-4,-5 system by means of multiplex polymerase chain reaction(PCR) technique. Methods HPA-2,-4,-5 antigen system genes were simultaneously amplified in one amplification system.The results of genotyping HPA-2,-4,-5 antigen system by multiplex PCR were compared with those obtained by PCR-sequence specific primer(SSP). Results The genotype of HPA-2,-4,-5 antigen system obtained by multiplex PCR in 75 healthy platelet donors were 70 of 2a/2a,5 of 2a/2b,0 of 2b/2b;73 of 4a/4a,2 of (4a/4b,) 0 of 4b/4b;66 of 5a/5a,9 of 5a/5b and 0 of 5b/5b.All were in agreement with those determined by PCR-SSP. Conclusions The method of multiplex PCR is convenient,economical,rapid,and accurate.It may be appropriate for genotyping of HPA.
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This study established a multiplex STR genotyping system through universal fluorescent primers in combination with sequencing.For each panel of multiplex STR genotyping amplification,two sets of primers were used,namely universal primers labeled with FAM and specific primers with an added 5' tail.We optimized the proportion of universal and specific primers and reaction of multiplex PCR was achieved.By using this method,five STR marker loci genotyping were successfully conducted within a single reaction.The optimized universal fluorescent primers amplification genotyping system can be used in multiplex STR genotyping,which allows reliable,effective and low-cost genotyping compared with regular microsatellite fluorescent detection assays.
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Background : Different polymerase chain reaction (PCR) techniques for human platelet antigens (HPA) genotyping have been implemented, in order to diagnose the clinical syndromes of patients with thrombocytopaenia and provide effective HPA‐matched platelet donors. Objectives : The aim of this study is to develop an in‐house multiplex PCR for HPA‐1 to ‐7 and ‐15 genotyping in the Thai population. Methods : One hundred DNA samples of known HPA genotyping by the PCR with sequence‐specific primers (PCR‐SSP), as previously described, were tested with the multiplex PCR. Additionally, 300 DNA samples of group O donors were tested for HPA‐1 to ‐7 and ‐15 genotyping using multiplex PCR. Results : The comparison of HPA‐1 to ‐7 and ‐15 genotype results between multiplex PCR and PCR‐SSP technique was in 100% concordance. Interestingly, HPA‐2b2b genotype was found in two samples; however, other low‐incidence genotypes such as HPA‐1b1b, HPA‐5b5b, HPA‐6b6b and HPA‐7b7b were not found in this study. Moreover, 30 samples were randomly tested twice for HPA genotyping using the multiplex PCR and demonstrated reproducible results. Conclusions : This study shows that the in‐house multiplex PCR is simple, cost‐effective and suitable for HPA genotyping for routine laboratories in other developing countries. Nevertheless, a large‐scale evaluation of this technique through multicentre analysis is suggested.
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[Objective] To develop a multiplex PCR-CE method for genotyping of human platelet antigens(HPA)-2,-3 and-15 and investigate gene frequencies of HPA-2,-3 and-15 in Dalian Han population with the method.[Methods] The allele-specific primers and common primers for genotyping of HPA-2,-3 and-15 were designed.The common primers were labeled with fluorescence.The genes of HPA-2,-3 and-15 were amplified using multiplex PCR.Then PCR products were detected using CE.HPAs were genotyped according to different length of PCR products.The multiplex PCR-CE was compared with the conventional PCR-SSP.In addition,dates of HPAs genotyping in Dalian Han population were compared with those in other Chinese populations.[Results] There was complete concordance of results for all samples tested by multiplex PCR-CE and PCR-SSP.The gene frequencies of HPA-2a,-2b,-3a,-3b,-15a and-15b in Dalian Han population were 0.9200,0.0800,0.5750,0.4250,0.5800 and 0.4200 respectively,which did not significantly differ from those in Shan dong,Shanghai and Zhejiang Han populations(P0.05).The gene frequencies of HPA-2,-3 in Dalian Han population did not significantly differ from those in Xinjiang Uighurs population(P0.05),but the gene frequency of HPA-15 in Dalian Han population significantly differed from that in Xinjiang Uighurs population(P0.05).[Conclusions] Multiplex PCR-CE for genotyping of HPAs is a fast,simple and accurate method.There is different in frequency distribution of HPA-15 alleles among different races of China.
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Targeted genotyping is an extremely powerful approach for the detection of known genetic variations that are biologically or clinically important. However, for non-model organisms, large-scale target genotyping in a cost-effective manner remains a major challenge. To address this issue, we present an ultrahigh-multiplex, in-solution probe array-based high-throughput diverse marker genotyping (HD-Marker) approach that is capable of targeted genotyping of up to 86 000 loci, with coverage of the whole gene repertoire, in what is a 27-fold and six-fold multiplex increase in comparison with the conventional Illumina GoldenGate and original HD-Marker assays, respectively. We perform extensive analyses of various ultrahigh-multiplex levels of HD-Marker (30 k-plex, 56 k-plex, and 86 k-plex) and show the power and excellent performance of the proposed method with an extremely high capture rate (about 96%) and genotyping accuracy (about 96%). With great advantages in terms of cost (as low as 0.0006 USD per genotype) and high technical flexibility, HD-Marker is a highly efficient and powerful tool with broad application potential for genetic, ecological, and evolutionary studies of non-model organisms.
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Objective To develop a method for rapid diagnosis and genotyping for Staphylococcus aureus and assess the genotyping efficiency and group distribution.Methods The assay of multiplex PCR was established for Staphylococcus aureus detecting and genotyping simultaneously.Both sensitivity and specificity of multiplex PCR were evaluated by colony forming unit(CFU) counting and gene sequencing,respectively.Results The sensitivity of multiplex PCR was 79 CFU/reaction;The genotyping efficiency(100%) by using the multiplex PCR was significantly higherthantheserotyping(79.78%)by serum agglutination reaction(χ2=15.4514,P0.01).The genotyping efficiency was 100% whereas the serotyping was 65.71% in the isolations from health carriers(χ2=8.661 3,P0.01).The sequencing results supported the genotyping ones among the samples in which the genotyping results conflicted to serotyping.Conclusion Both sensitivity and specificity by employing the multiplex PCR were higher than those by serum agglurination reaction inthe Staphylococcus aureus identification and group-typing.The genotyping was superior to serotyp ing for grouping efficacy.
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Different polymerase chain reaction (PCR) techniques for human neutrophil antigens (HNA) genotyping have been implemented to diagnose the clinical conditions of patients with alloimmune neutropenia, febrile non-hemolytic transfusion reactions, and transfusion-related acute lung injury and to provide effective HNAmatched granulocyte transfusions. The present study aimed to develop an in-house multiplex-PCR for HNA-1, -3, -4, and -5 genotyping in the Thai population.Altogether, 500 DNA samples obtained from unrelated, healthy Thai blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok, Thailand were included. Three hundred DNA samples of known HNA genotyping based on PCR with sequence specific primers (PCR-SSP), as previously described, were tested with the newly developed multiplex PCR. Additionally, 200 DNA samples of unknown HNA genotyped donors were tested for HNA-1, -3, -4, and -5 genotyping using multiplex-PCR.Validity of HNA-1, -3, -4, and -5 genotyping by multiplex PCR using known DNA controls and the comparison of the genotyping results between PCR-SSP and multiplex PCR were in agreement. Interestingly, the rare genotype HNA-4b4b was not found in this study, similar to previous studies in Thai and other populations. Moreover, 30 samples were randomly tested twice for HNA genotyping using the multiplex-PCR and demonstrated reproducible results and were confirmed by DNA sequencing.This study shows that the newly developed multiplex-PCR is cost effective and less time consuming compared with PCR-SSP. The multiplex PCR can be used as an alternative technique for HNA-1, -3, -4, and -5 genotyping for routine testing, especially in other developing countries due to its simplicity and accuracy.
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Objective To develop a method for genotyping of the human platelet antigen (HPA)-2,-4,-5 system by means of multiplex polymerase chain reaction (PCR) technique Methods HPA-2,-4,-5 antigen system genes were simultaneously amplified in one amplification system. The results of genotyping HPA-2,-4,-5 antigen system by multiplex PCR were compared with those obtained by PCR-sequence specific primer (SSP). Results The genotype of HPA-2,-4,-5 antigen system obtained by multiplex PCR in 75 healthy platelet donors were 70 of 2a/2a, 5 of 2a/2b, 0 of 2b/2b; 73 of 4a/4a, 2 of 4a/4b, 0 of 4b/4b; 66 of 5a/5a, 9 of 5a/5b and 0 of 5b/5b. All were in agreement with those determined by PCR-SSP. Conclusions The method of multiplex PCR is convenient, economical, rapid, and accurate. It may be appropriate for genotyping of HPA.
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