Rapid lactic acid bacteria identification in dairy products by high-resolution melt analysis of DGGE bands

Aim: To investigate the application of high‐resolution melt (HRM) analysis for rapid species‐level identification of lactic acid bacteria (LAB) communities in dairy products, as well as for bacterial community profiling and monitoring. Methods and Results: First, comparisons of HRM profiles of known reference strains of LAB and their denaturing gradient gel electrophoresis (DGGE) bands showed very good agreement, allowing species recognition and identification from DGGE bands by HRM. Second, samples of cheese, kefir grains and kefir were characterized by PCR‐DGGE, and melting profiles of DGGE bands were compared with known reference strains. Of the 13 DGGE bands, ten were identified by HRM by comparison with the reference strains and only three required sequencing for identification. Use of HRM profiling for comparison and monitoring of total LAB communities from dairy products or starter cultures was also evaluated, and good agreement was found when comparing clustering of DGGE band profiles with clustering of HRM melting profiles. Conclusion: Identification of DGGE bands is possible by comparison of HRM melting profiles with known reference strains. Significance and Impact of the Study: HRM profiling is suggested as an additional approach for identification of DGGE bands.     

Application of high-resolution melting for genotyping bovine mitochondrial DNA

Recent studies have demonstrated that mitochondrial DNA (mtDNA) haplotype has a significant impact on the efficiency of bovine somatic cell nuclear transfer. Conventional methods for detecting mtDNA variations and haplotypes, such as restriction fragment length polymorphism (RFLP), temporal temperature gradient gel electrophoresis, dHPLC and sequencing, are labor intensive or expensive and have low sensitivity. High-resolution melting (HRM) analysis is a new technique for mutation detection and has the advantages of speed, cost, and accuracy. Here, we describe the genotyping of bovine mtDNA using HRM analysis. DNA samples containing mtDNA were extracted from 75 Holstein cows and subjected to rapid-cycle (<20 min) PCR of small amplicons (<120 bp) using specific primer sets. Capillaries containing the PCR products were then subjected to HRM analysis; data were acquired in 2 min and analyzed using the instrument's software. Five common bovine mtDNA single nucleotide polymorphisms were identified: 9602 G>A, 169 A>G, 166A>G with 173A>G, and 363C>G. These results agree with both sequencing and RFLP analysis. In addition, a very small amount of heteroplasmic variants (<5%) was sufficiently to be distinguished by HRM analysis that would be very useful to differentiate heteroplasmy vs. homoplasmy. HRM analysis thus provides a new approach to genotyping bovine mtDNA sequence variations and has many advantages over other methods, including speed of analysis, cost, and accuracy. We believe this will be a valuable technique for determining the efficiency of nuclear transfer in cloned embryos and for studying maternal effects on nuclear-cytoplasm interactions.     

Denaturing Gradient Gel Electrophoresis Can Rapidly Display the Bacterial Diversity Contained in 16S rDNA Clone Libraries

Two different strategies for molecular analysis of bacterial diversity, 16S rDNA cloning and denaturing gradient gel electrophoresis (DGGE), were combined into a single protocol that took advantage of the best attributes of each: the ability of cloning to package DNA sequence information and the ability of DGGE to display a community profile. In this combined protocol, polymerase chain reaction products from environmental DNA were cloned, and then DGGE was used to screen the clone libraries. Both individual clones and pools of randomly selected clones were analyzed by DGGE, and these migration patterns were compared to the conventional DGGE profile produced directly from environmental DNA. For two simple bacterial communities (biofilm from a humics-fed laboratory reactor and planktonic bacteria filtered from an urban freshwater pond), pools of 35–50 clones produced DGGE profiles that contained most of the bands visible in the conventional DGGE profiles, indicating that the clone pools were adequate for identifying the dominant genotypes. However, DGGE profiles of two different pools of 50 clones from a lawn soil clone library were distinctly different from each other and from the conventional DGGE profile, indicating that this small number of clones poorly represented the bacterial diversity in soil. Individual clones with the same apparent DGGE mobility as prominent bands in the humics reactor community profiles were sequenced from the clone plasmid DNA rather than from bands excised from the gel. Because a longer fragment was cloned (∼1500 bp) than was actually analyzed in DGGE (∼350 bp), far more sequence information was available using this approach that could have been recovered from an excised gel band. This clone/DGGE protocol permitted rapid analysis of the microbial diversity in the two moderately complex systems, but was limited in its ability to represent the diversity in the soil microbial community. Nonetheless, clone/DGGE is a promising strategy for fractionating diverse microbial communities into manageable subsets consisting of small pools of clones.     

Distinguishing Anuran species by high‐resolution melting analysis of the COI barcode (COI‐HRM)

Taxonomic identification can be difficult when two or more species appear morphologically similar. DNA barcoding based on the sequence of the mitochondrial cytochrome c oxidase 1 gene (COI) is now widely used in identifying animal species. High‐resolution melting analysis (HRM) provides an alternative method for detecting sequence variations among amplicons without having to perform DNA sequencing. The purpose of this study was to determine whether HRM of the COI barcode can be used to distinguish animal species. Using anurans as a model, we found distinct COI melting profiles among three congeners of both Lithobates spp. and Hyla spp. Sequence variations within species shifted the melting temperature of one or more melting domains slightly but do not affect the distinctness of the melting profiles for each species. An NMDS ordination plot comparing melting peak profiles among eight Anuran species showed overlapping profiles for Lithobates sphenocephala and Gastrophryne carolinensis. The COI amplicon for both species contained two melting domains with melting temperatures that were similar between the two species. The two species belong to two different families, highlighting the fact that COI melting profiles do not reveal phylogenetic relationships but simply reflect DNA sequence differences among stretches of DNA within amplicons. This study suggests that high‐resolution melting analysis of COI barcodes (COI‐HRM) may be useful as a simple and rapid method to distinguish animal species that appear morphologically similar.     

Analysis of HIV Using a High Resolution Melting (HRM) Diversity Assay: Automation of HRM Data Analysis Enhances the Utility of the Assay for Analysis of HIV Incidence

Background

HIV diversity may be a useful biomarker for discriminating between recent and non-recent HIV infection. The high resolution melting (HRM) diversity assay was developed to quantify HIV diversity in viral populations without sequencing. In this assay, HIV diversity is expressed as a single numeric HRM score that represents the width of a melting peak. HRM scores are highly associated with diversity measures obtained with next generation sequencing. In this report, a software package, the HRM Diversity Assay Analysis Tool (DivMelt), was developed to automate calculation of HRM scores from melting curve data.

Methods

DivMelt uses computational algorithms to calculate HRM scores by identifying the start (T1) and end (T2) melting temperatures for a DNA sample and subtracting them (T2–T1 = HRM score). DivMelt contains many user-supplied analysis parameters to allow analyses to be tailored to different contexts. DivMelt analysis options were optimized to discriminate between recent and non-recent HIV infection and to maximize HRM score reproducibility. HRM scores calculated using DivMelt were compared to HRM scores obtained using a manual method that is based on visual inspection of DNA melting curves.

Results

HRM scores generated with DivMelt agreed with manually generated HRM scores obtained from the same DNA melting data. Optimal parameters for discriminating between recent and non-recent HIV infection were identified. DivMelt provided greater discrimination between recent and non-recent HIV infection than the manual method.

Conclusion

DivMelt provides a rapid, accurate method of determining HRM scores from melting curve data, facilitating use of the HRM diversity assay for large-scale studies.     

Detection of base mutations in genomic DNA using denaturing gradient gel electrophoresis (DGGE) followed by transfer and hybridization with gene-specific probes

It has been shown that minor differences, such as single-base-pair substitutions between otherwise identical DNA fragments can result in altered melting behavior detectable by denaturing gradient gel electrophoresis (DGGE). Sequence variations in only a small DNA region within one locus can be detected using the previously described procedures. We have developed a method for the efficient Southern transfer of genomic DNA fragments from the denaturing gradient gels in order to be able to analyze larger regions in several loci for variation. The gels were made using polyacrylamide containing 2% low-geling-temperature agarose (LGT). The polyacrylamide gel (PAG) was crosslinked with a reversible crosslinker, and after electrophoresis the crosslinks were cleaved, the structure of the gel being maintained by the agarose. After this treatment of the denaturing gels, more than 90% of the DNA fragments could be transferred to nylon membranes by alkaline transfer, while electroblotting transferred only 10% of the DNA. Hybridization with gene-specific probes was then performed. We have used this technique to identify an RFLP in the COL1A2 gene in a human genomic DNA sample. The transfer technique described should make the use of DGGE more widely applicable since the genomic DNA fragments separated on one gel can be screened with several different probes, both cDNA and genomic probes.     

Recognizing diversity in coral symbiotic dinoflagellate communities

A detailed understanding of how diversity in endosymbiotic dinoflagellate communities maps onto the physiological range of coral hosts is critical to predicting how coral reef ecosystems will respond to climate change. Species-level taxonomy of the dinoflagellate genus Symbiodinium has been predominantly examined using the internal transcribed spacer (ITS) region of the nuclear ribosomal array (rDNA ITS2) and downstream screening for dominant types using denaturing gradient gel electrophoresis (DGGE). Here, ITS2 diversity in the communities of Symbiodinium harboured by two Hawaiian coral species was explored using direct sequencing of clone libraries. We resolved sixfold to eightfold greater diversity per coral species than previously reported, the majority of which corresponds to a novel and distinct phylogenetic lineage. We evaluated how these sequences migrate in DGGE and demonstrate that this method does not effectively resolve this diversity. We conclude that the Porites spp. examined here harbour diverse assemblages of novel Symbiodinium types and that cloning and sequencing is an effective methodological approach for resolving the complexity of endosymbiotic dinoflagellate communities harboured by reef corals.     

变性梯度凝胶电泳(DGGE)在微生物生态学中的应用

由于从环境样品中分离和培养细菌的困难,分子生物学方法已发展用来描述和鉴定微生物群落。近年来基于DNA方法的群落分析得到了迅速的发展,如PCR扩增技术,克隆文库法,荧光原位杂交法,限制性酶切片段长度多态性法,变性和温度梯度凝胶电泳法。DGGE已广泛用于分析自然环境中细菌、蓝细菌,古菌、微微型真核生物、真核生物和病毒群落的生物多样性。这一技术能够提供群落中优势种类信息和同时分析多个样品。具有可重复和容易操作等特点,适合于调查种群的时空变化,并且可通过对切下的带进行序列分析或与特异性探针杂交分析鉴定群落成员。DGGE分析微生物群落的一般步骤如下：一是核酸的提取,二是16S rRNA,18S rRNA或功能基因如可容性甲烷加单氧酶羟化酶基因(mmoX)和氨加单氧酶a一亚单位基因(amoA)片段的扩增,三是通过DGGE分析PCR产物。DGGE使用具有化学变性剂梯度的聚丙烯酰胺凝胶,该凝胶能够有区别的解链PCR扩增产物。由PCR产生的不同的DNA片段长度相同但核苷酸序列不同。因此不同的双链DNA片段由于沿着化学梯度的不同解链行为将在凝胶的不同位置上停止迁移。DNA解链行为的不同导致一个凝胶带图案,该图案是微生物群落中主要种类的一个轮廓。DGGE使用所有生物中保守的基因片段如细菌中的16S rRNA基因片段和真菌中的18S rRNA基因片段。然而同其他分子生物学方法一样,DGGE也有缺陷,其中之一是只能分离较小的片段,使用于系统发育分析比较和探针设计的序列信息量受到了限制。在某些情况下,由于所用基因的多拷贝导致一个种类多于一条带,因此不易鉴定群落结构到种的水平。此外,该技术具有内在的如单一细菌种类16S rDNA拷贝之间的异质性问题,可导致自然群落中微生物数量的过多估计。DGGE是分析微生物群落的一种有力的工具。不过为了减少DGGE和其它技术的缺陷,建议研究者结合DGGE和其它分子及微生物学方法以便更详细的观察微生物的群落结构和功能。     

Comprehensive, rapid and sensitive detection of sequence variants of human mitochondrial tRNA genes.

In the present study, a comprehensive, rapid and sensitive method for screening sequence variation of the human mitochondrial tRNA genes has been developed. For this purpose, the denaturing gradient gel electrophoresis (DGGE) technique has been appropriately modified for simultaneous mutation analysis of a large number of samples and adapted so as to circumvent the problems caused by the anomalous electrophoretic behavior of DNA fragments encoding tRNA genes. Eighteen segments of mitochondrial DNA (mtDNA), each containing a single uniform melting domain, were selected to cover all tRNA-encoding regions using the computer program MELT94. All 18 segments were simultaneously analyzed by electrophoresis through a single broad range denaturing gradient gel under rigorously defined conditions, which prevent band broadening and other migration abnormalities from interfering with detection of sequence variants. All base substitutions tested, which include six natural mutations and 14 artificially introduced ones, have been detected successfully in the present study. Several types of evidence strongly suggest that the anomalous behavior in DGGE of tRNA gene-containing mtDNA fragments reflects their tendency to form temporary or stable alternative secondary structures under semi-denaturing conditions. The high sensitivity of the method, which can detect as low as 10% of mutant mtDNA visually, makes it valuable for the analysis of heteroplasmic mutations.     

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

Unexpected behavior of H2Kb mutant DNAs in denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) is based upon the different melting behaviors of DNA molecules in a chemical denaturant gradient according to their sequences. This technique has recently become a widespread tool to detect mutations. The introduction of a GC-clamp enables the detection of most single base differences between two DNA molecules. As a test system we have applied the polymerase chain reaction (PCR) in combination with DGGE to detect a number of mutations in the mouse H2Kb DNA sequence. A wide variety of spontaneous in vivo mutations of this haplotype have been reported in the C57BL/6J mouse strain and are clustered in the alpha 1 and alpha 2 domains. The combination of PCR and DGGE revealed almost all base changes present in the H2Kb mutants used. However, most of the PCR products of these mutants showed melting behavior which is not easily predicted. We suggest that in addition to current simple theory, which considers that the migration of a DNA molecule in a denaturing gradient depends primarily on its initial melting behavior, additional factors such as secondary structure in partially melted molecules may play a role and can be used to detect mutations.     

“Blind” mapping of genic DNA sequence polymorphisms in Lolium perenne L. by high resolution melting curve analysis

High resolution melting curve analysis (HRM) measures dissociation of double stranded DNA of a PCR product amplified in the presence of a saturating fluorescence dye. Recently, HRM proved successful to genotype DNA sequence polymorphisms such as SSRs and SNPs based on the shape of the melting curves. In this study, HRM was used for simultaneous screening and genotyping of genic DNA sequence polymorphisms identified in the Lolium perenne F2 mapping population VrnA. Melting profiles of PCR products amplified from previously published gene loci and from a novel gene putatively involved in vernalization response successfully discriminated genotypes in absence of allelic sequence information, and allowed to determine allele segregation in VrnA. Here we introduce the concept of “blind” mapping based on HRM as a powerful, fast and cheap method to map any DNA sequence polymorphisms without prior knowledge of allelic sequences in the key grassland species perennial ryegrass (Lolium perenne L.).     

Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation

In this article, we show that high resolution melting analysis (HRM) is a sensitive and specific method for the detection of methylation. Methylated DNA and unmethylated DNA acquire different sequences after bisulphite treatment resulting in PCR products with markedly different melting profiles. We used PCR to amplify both methylated and unmethylated sequences and assessed HRM for the determination of the methylation status of the MGMT promoter region. Reconstruction experiments showed that MGMT methylation could be detected at levels as low as 0.1%. Moreover, MS-HRM allows for estimation of the methylation level by comparing the melting profiles of unknown PCR products to the melting profiles of PCR products derived from standards with a known unmethylated to methylated template ratio. We used MS-HRM for the analysis of eight cell lines of known methylation status and a panel of colorectal cancer specimens. The simplicity and high reproducibility of the MS-HRM protocol makes MS-HRM the method of choice for methylation assessment in many diagnostic and research applications.     

应用HRM技术对CYP2C19*2和CYP2C19*3进行双重SNP分型

氯吡格雷是一种广泛用于预防静脉血栓形成的抗血小板药物。研究表明, 携带有CYP2C19基因功能缺失型等位基因CYP2C19*2、CYP2C19*3的病人, 其体内代谢氯吡格雷成为其活性形式的能力降低, 导致氯吡格雷抑制血小板聚集功能减弱。文章旨在建立一种利用高分辨率熔解曲线分析(High-resolution melting curve analysis,HRM)技术在闭合单管中同时对CYP2C19*2、CYP2C19*3两个多态性位点进行简便、准确分型的方法。本实验针对两个SNP位点分别设计特异性的HRM引物, 并在两个位点引物的5′端分别加上富含AT和GC的序列, 保证两个位点的扩增产物熔解峰无重叠。利用HRM技术, 快速、灵敏地对64例随机DNA样本的CYP2C19*2 、CYP2C19*3两个多态性位点进行了基因分型, 且HRM方法的分型结果与测序验证结果完全一致。因此, 利用HRM技术可以实现在闭合单管中简便、准确地对CYP2C19*2 、CYP2C19*3两个多态性位点同时进行基因分型。该方法有望应用于临床, 指导氯吡格雷的个体化用药。     

Biogeography of two species of Symbiodinium (Freudenthal) inhabiting the intertidal sea anemone Anthopleura elegantissima (Brandt)

We have analyzed the genetic profiles of dinoflagellate populations obtained from the Pacific coast sea anemone Anthopleura elegantissima (Brandt) at collection sites from Washington to California. Genetic differences within the symbiont populations of California anemones have been uncovered by restriction length polymorphism (RFLP) analysis of the small subunit (SSU) and large subunit (LSU) ribosomal RNA genes, and by denaturing gradient gel electrophoresis (DGGE) of the internal transcribed spacer region 2 (ITS 2). The existence of two Symbiodinium species is substantiated by sequence analysis of the variable regions V1, V2, and V3 of the SSUrDNA, which also establishes their phylogenetic relatedness to other members of the genus Symbiodinium. Anemones from Washington and Oregon harbor a single dinoflagellate species, for which we propose the name S. muscatinei sp. nov. At these northern locations, S. muscatinei either exists alone or co-occurs with the Chlorella-like green algal symbiont. Our results indicate that S. muscatinei co-occurs with a second dinoflagellate, S. californium, in mixed populations in central and southern California. We suggest that the geographic distribution of these dinoflagellates is related to the temperature cline created by latitude.     

A New Framework to Accurately Quantify Soil Bacterial Community Diversity from DGGE

Denaturing gradient gel electrophoresis (DGGE) has been and remains extensively used to assess and monitor the effects of various treatments on soil bacterial communities. Considering only abundant phylotypes, the diversity estimates produced by this technique have been proven to be uncorrelated to true community diversity. The aim of this paper was to develop a framework to estimate a community’s true diversity from DGGE. Developed using in silico DGGE profiles generated from published pyrosequencing datasets, this framework elongates the rank-abundance distributions (RADs) drawn by band quantification using the peak-to-signal ratio (PSR) parameter, which was proven to be related to bacterial richness. The ability to compare DGGE-based diversity estimates to the true diversity of communities led to a unique opportunity to identify potential pitfalls when analyzing DGGE gels with commercial analysis software programs and gain insight into the process of DNA band clustering in the profiles. Bacterial diversity was compared through richness, Shannon, and Simpson’s 1/D indices. Intermediate results demonstrated that, even though commercial gel analysis software programs were unable to produce consistent results throughout all samples, a newly developed Matlab-based framework unraveled the dominance profiles of communities from band quantification. Elongating these partial RADs using the PSRs extracted from the DGGE profiles chiefly made it possible to accurately estimate the true diversity of communities. For all the samples analyzed, the estimated Shannon and Simpson’s 1/D were accurate at ±10 %. Richness estimations were less accurate, ranging from ?11 to 31 % of the expected values. The framework showed great potential to study the structure and diversity of soil bacterial communities.     

Resolution of a missense mutant in human genomic DNA by denaturing gradient gel electrophoresis and direct sequencing using in vitro DNA amplification: HPRT Munich.

The combination of denaturing gradient gel electrophoresis (DGGE) and in vitro DNA amplification has allowed us to (1) localize a DNA mutation to a given 100-bp region of the human genome and (2) rapidly sequence the DNA without cloning. DGGE showed that a mutation had occurred, but the technique revealed little about the nature or position of that mutation. The region of the genome containing the mutation was amplified by the polymerase chain-reaction technique, providing DNA of sufficient quality and quantity for direct sequencing. Amplification was performed with a 32P end-labeled primer that allowed direct Maxam-Gilbert sequencing of the amplified product without cloning. HPRTMunich was found to contain a single-base-pair substitution, a C-to-A transversion at base-pair position 397. We report the generation of a 169-bp, wild-type DNA probe that encompasses most of exon 3 of the human hypoxanthine guanine phosphoribosyltransferase (HPRT) gene and contains a low-temperature melting domain of approximately 100 bp. HPRTMunich, an HPRT mutant isolated from a patient with gout, has a single amino acid substitution; the corresponding DNA sequence alteration must lie within the low-temperature melting domain of exon 3. We report the separation of HPRTMunich from the wild-type sequence using DGGE. In addition to base-pair substitutions, DGGE is also sensitive to the methylation state of the molecule. The cDNA for HPRT was cloned into a vector and propagated in Escherichia coli dam+ and dam- strains; thus, methylated and unmethylated HPRT cDNA was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of sequence alterations in a defined DNA region: comparison of temperature-modulated heteroduplex analysis and denaturing gradient gel electrophoresis

The ability to detect DNA sequence heterogeneity quickly and reliably is becoming increasingly important as more genes involved in disease processes are discovered. We have assessed the ability of a high pressure liquid chromatography technique (HPLC) termed temperature-modulated heteroduplex analysis (TMHA) to detect a collection of 20 point mutations distributed throughout a 279 base pair fragment spanning the exon 8 region of the human HPRT gene. All mutant/wild type heteroduplexes formed from mutations in the lowest temperature melting domain of the fragment were easily resolved from the corresponding mutant and wt homoduplexes, while those generated from mutants in the next higher melting domain barely resolved from their parental homoduplexes. For comparison, identical heteroduplex samples were subjected to denaturing gradient gel electrophoresis (DGGE). Heteroduplexes in the lowest temperature melting domain were easily resolved, while no resolution was achieved with those in the next higher melting domain. These results suggest that TMHA and DGGE are measuring similar melting characteristics in heteroduplex molecules. TMHA appears to be a robust approach for detecting and/or purifying a wide variety of mutations in a defined region of DNA, provided that the melting characteristics of the fragment under study are carefully considered.     

Detection of mutations in GC-rich DNA by bisulphite denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) in combination with PCR and 'GC-clamping' has proven highly efficient as a method for detection of DNA sequence differences. Due to strand dissociation phenomena, however, its use has been limited to the analysis of sequences with a relatively low content of GC pairs. This paper describes how treatment of template DNA with sodium bisulphite drastically lowers the melting temperature of very GC-rich sequences and renders them amenable to DGGE analysis. We demonstrate the use of bisulphite DGGE for rapid and efficient detection of mutations in the p16(INK4/CDKN2) tumour suppressor gene.     

High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.