High-resolution DNA melt curve analysis of the clustered, regularly interspaced short-palindromic-repeat locus of Campylobacter jejuni

A novel method for genotyping the clustered, regularly interspaced short-palindromic-repeat (CRISPR) locus of Campylobacter jejuni is described. Following real-time PCR, CRISPR products were subjected to high-resolution melt (HRM) analysis, a new technology that allows precise melt profile determination of amplicons. This investigation shows that the CRISPR HRM assay provides a powerful addition to existing C. jejuni genotyping methods and emphasizes the potential of HRM for genotyping short sequence repeats in other species.     

Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis

Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.     

Trainable High Resolution Melt Curve Machine Learning Classifier for Large-Scale Reliable Genotyping of Sequence Variants

High resolution melt (HRM) is gaining considerable popularity as a simple and robust method for genotyping sequence variants. However, accurate genotyping of an unknown sample for which a large number of possible variants may exist will require an automated HRM curve identification method capable of comparing unknowns against a large cohort of known sequence variants. Herein, we describe a new method for automated HRM curve classification based on machine learning methods and learned tolerance for reaction condition deviations. We tested this method in silico through multiple cross-validations using curves generated from 9 different simulated experimental conditions to classify 92 known serotypes of Streptococcus pneumoniae and demonstrated over 99% accuracy with 8 training curves per serotype. In vitro verification of the algorithm was tested using sequence variants of a cancer-related gene and demonstrated 100% accuracy with 3 training curves per sequence variant. The machine learning algorithm enabled reliable, scalable, and automated HRM genotyping analysis with broad potential clinical and epidemiological applications.     

Fingerprinting of Campylobacter jejuni by Using Resolution-Optimized Binary Gene Targets Derived from Comparative Genome Hybridization Studies

The aim of this investigation was to exploit the vast comparative data generated by comparative genome hybridization (CGH) studies of Campylobacter jejuni in developing a genotyping method. We examined genes in C. jejuni that exhibit binary status (present or absent between strains) within known plasticity regions, in order to identify a minimal subset of gene targets that provide high-resolution genetic fingerprints. Using CGH data from three studies as input, binary gene sets were identified with “Minimum SNPs” software. “Minimum SNPs” selects for the minimum number of targets required to obtain a predefined resolution, based on Simpson's index of diversity (D). After implementation of stringent criteria for gene presence/absence, eight binary genes were found that provided 100% resolution (D = 1) of 20 C. jejuni strains. A real-time PCR assay was developed and tested on 181 C. jejuni and Campylobacter coli isolates, a subset of which have previously been characterized by multilocus sequence typing, flaA short variable region sequencing, and pulsed-field gel electrophoresis. In addition to the binary gene real-time PCR assay, we refined the seven-member single nucleotide polymorphism (SNP) real-time PCR assay previously described for C. jejuni and C. coli. By normalizing the SNP assay with the respective C. jejuni and C. coli ubiquitous genes, mapA and ceuE, the polymorphisms at each SNP could be determined without separate reactions for every polymorphism. We have developed and refined a rapid, highly discriminatory genotyping method for C. jejuni and C. coli that uses generic technology and is amenable to high-throughput analyses.     

空肠弯曲菌CRISPR/Cas系统的研究进展

多数细菌都存在发挥免疫防御机制作用的CRISPR/Cas系统,在不同种属间呈现多态性。空肠弯曲菌是全球范围内重要的食源性病原菌,所致疾病也是典型的自限性疾病,其复杂的致病机制并未得到明确的解析,而空肠弯曲菌CRISPR/Cas系统的结构呈现多态性,研究两者关系仍存在诸多限制。本文从CRISPR/Cas系统在空肠弯曲菌中的结构、机制及技术应用等方面的研究进展进行综述,为探索空肠弯曲菌致病机制提供新思路。     

Automated Classification and Cluster Visualization of Genotypes Derived from High Resolution Melt Curves

Introduction

High Resolution Melting (HRM) following PCR has been used to identify DNA genotypes. Fluorescent dyes bounded to double strand DNA lose their fluorescence with increasing temperature, yielding different signatures for different genotypes. Recent software tools have been made available to aid in the distinction of different genotypes, but they are not fully automated, used only for research purposes, or require some level of interaction or confirmation from an analyst.

Materials and Methods

We describe a fully automated machine learning software algorithm that classifies unknown genotypes. Dynamic melt curves are transformed to multidimensional clusters of points whereby a training set is used to establish the distribution of genotype clusters. Subsequently, probabilistic and statistical methods were used to classify the genotypes of unknown DNA samples on 4 different assays (40 VKORC1, CYP2C9*2, CYP2C9*3 samples in triplicate, and 49 MTHFR c.665C>T samples in triplicate) run on the Roche LC480. Melt curves of each of the triplicates were genotyped separately.

Results

Automated genotyping called 100% of VKORC1, CYP2C9*3 and MTHFR c.665C>T samples correctly. 97.5% of CYP2C9*2 melt curves were genotyped correctly with the remaining 2.5% given a no call due to the inability to decipher 3 melt curves in close proximity as either homozygous mutant or wild-type with greater than 99.5% posterior probability.

Conclusions

We demonstrate the ability to fully automate DNA genotyping from HRM curves systematically and accurately without requiring any user interpretation or interaction with the data. Visualization of genotype clusters and quantification of the expected misclassification rate is also available to provide feedback to assay scientists and engineers as changes are made to the assay or instrument.     

Genotyping of the porcine ryanodine receptor 1 (RYR1) and estrogen receptor 1 (ESR1) genes by high resolution melting (HRM) approach

During the last decade, DNA mutations in the porcine ryanodine receptor 1 gene (RYR1, C1843T) and the estrogen receptor 1 gene (ESR1, T1665G), have been widely used in marker-assisted selection (MAS) for the pig industry. These 2 well-characterized SNPs in RYR1 and ESR1 are responsible for porcine stress syndrome (PSS) and litter size, respectively. Here, we describe a reliable, high-efficiency method for the genotyping of these 2 genes using the high-resolution melting (HRM) method. The HRM approach exhibited high-accuracy and repeatability, comparable with the classic PCR-restriction fragment length polymorphism (PCR-RFLP) approach, and is potentially suitable for large-scale genotyping in commercial pig farms.     

Lactobacillus buchneri Genotyping on the Basis of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Locus Diversity

Clustered regularly interspaced short palindromic repeats (CRISPR) in combination with associated sequences (cas) constitute the CRISPR-Cas immune system, which uptakes DNA from invasive genetic elements as novel “spacers” that provide a genetic record of immunization events. We investigated the potential of CRISPR-based genotyping of Lactobacillus buchneri, a species relevant for commercial silage, bioethanol, and vegetable fermentations. Upon investigating the occurrence and diversity of CRISPR-Cas systems in Lactobacillus buchneri genomes, we observed a ubiquitous occurrence of CRISPR arrays containing a 36-nucleotide (nt) type II-A CRISPR locus adjacent to four cas genes, including the universal cas1 and cas2 genes and the type II signature gene cas9. Comparative analysis of CRISPR spacer content in 26 L. buchneri pickle fermentation isolates associated with spoilage revealed 10 unique locus genotypes that contained between 9 and 29 variable spacers. We observed a set of conserved spacers at the ancestral end, reflecting a common origin, as well as leader-end polymorphisms, reflecting recent divergence. Some of these spacers showed perfect identity with phage sequences, and many spacers showed homology to Lactobacillus plasmid sequences. Following a comparative analysis of sequences immediately flanking protospacers that matched CRISPR spacers, we identified a novel putative protospacer-adjacent motif (PAM), 5′-AAAA-3′. Overall, these findings suggest that type II-A CRISPR-Cas systems are valuable for genotyping of L. buchneri.     

High-Resolution Genotyping of Campylobacter Strains Isolated from Poultry and Humans with Amplified Fragment Length Polymorphism Fingerprinting

For epidemiological studies of Campylobacter infections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing of Campylobacter jejuni and Campylobacter coli strains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleases HindIII and HhaI were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amplified fragments ranging from 50 to 500 bp long. The discriminatory power of AFLP was assessed with a C. jejuni strain, an isogenic flagellin mutant, and distinct C. jejuni strains having known pulsed-field gel electrophoresis and fla PCR-restriction fragment length polymorphism genotypes. Unrelated C. jejuni strains produced heterogeneous patterns, whereas genetically related strains produced similar AFLP patterns. Twenty-five Campylobacter strains obtained from poultry farms in The Netherlands grouped in three C. jejuni clusters that were separate from a C. coli cluster. The band patterns of 10 C. jejuni strains isolated from humans were heterogeneous, and most of these strains grouped with poultry strains. Our results show that AFLP analysis can distinguish genetically unrelated strains from genetically related strains of Campylobacter species. However, desirable genetically related strains can be differentiated by using other genotyping methods. We concluded that automated AFLP analysis is an attractive tool which can be used as a primary method for subtyping large numbers of Campylobacter strains and is extremely useful for epidemiological investigations.     

Evaluation of High Resolution Melting for MTHFR C677T Genotyping in Congenital Heart Disease

Background

High resolution melting (HRM) is a simple, flexible and low-cost mutation screening technique. The methylenetetrahydrofolate reductase (MTHFR) gene encoding a critical enzyme, potentially affects susceptibility to some congenital defects like congenital heart disease (CHD). We evaluate the performance of HRM for genotyping of the MTHFR gene C677T locus in CHD cases and healthy controls of Chinese Han population.

Methods

A total of 315 blood samples from 147 CHD patients (male72, female 75) and 168 healthy controls (male 92, female 76) were enrolled in the study. HRM was utilized to genotype MTHFR C677T locus of all the samples. The results were compared to that of PCR-RFLP and Sanger sequencing. The association of the MTHFR C677T genotypes and the risk of CHD was analyzed using odds ratio with their 95% confidence interval (CIs) from unconditional logistic regression.

Results

All the samples were successfully genotyped by HRM within 1 hour and 30 minutes while at least 6 hours were needed for PCR-RFLP and sequencing. The genotypes of MTHFR C677T CC, CT, and TT were 9.52%, 49.66%, and 40.82% in CHD group but 29.17%, 50% and 20.83% in control group, which were identical using both methods of HRM and PCR-RFLP, demonstrating the sensitivity and specificity of HRM were all 100%.

Conclusion

MTHFR C677T is a potential risk factor for CHD in our local residents of Shandong province in China. HRM is a fast, sensitive, specific and reliable method for clinical application of genotyping.     

High-resolution melting analysis for genotyping of the myotonic dystrophy type 1 associated Alu insertion/deletion polymorphism

Since its introduction, high-resolution melting (HRM) analysis has been used for genotyping of various types of sequence alterations. In this study, we report the use of HRM for genotyping of the 1-kb insertion/deletion polymorphism, involving a problematic region of five consecutive Alu elements, that is associated with myotonic dystrophy type 1. We combined a three-primer polymerase chain reaction (PCR) amplification approach with HRM using two primer sets. Analyses based on curve shapes are sensitive enough to differentiate between genotypes with both primer sets. In addition, the newly designed insertion-specific primer from the second primer set equalizes the allele-specific amplicon lengths, thereby reducing the possibility of preferential amplification of shorter fragments.     

Amplified Fragment Length Polymorphism Analysis of Campylobacter jejuni Strains Isolated from Chickens and from Patients with Gastroenteritis or Guillain-Barré or Miller Fisher Syndrome

The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships between Campylobacter jejuni strains infecting chickens (n = 54) and those causing gastroenteritis in humans (n = 53). In addition, C. jejuni strains associated with the development of Guillain-Barré syndrome (GBS) (n = 14) and Miller Fisher syndrome (MFS) (n = 4), two related acute paralytic syndromes in human, were included. Strains were isolated between 1989 and 1998 in The Netherlands. The AFLP banding patterns were analyzed with correlation-based and band-based similarity coefficients and UPGMA (unweighted pair group method using average linkages) cluster analysis. All C. jejuni strains showed highly heterogeneous fingerprints, and no fingerprints exclusive for chicken strains or for human strains were obtained. All strains were separated in two distinct genetic groups. In group A the percentage of human strains was significantly higher and may be an indication that genotypes of this group are more frequently associated with human diseases. We conclude that C. jejuni from chickens cannot be distinguished from human strains and that GBS or MFS related strains do not belong to a distinct genetic group.     

Comparative Genotyping of Campylobacter jejuni Strains from Patients with Guillain-Barré Syndrome in Bangladesh

Background

Campylobacter jejuni is a common cause of acute gastroenteritis and is associated with post-infectious neuropathies such as the Guillain-Barré syndrome (GBS) and the Miller Fisher syndrome (MFS). We here present comparative genotyping of 49 C. jejuni strains from Bangladesh that were recovered from patients with enteritis or GBS. All strains were serotyped and analyzed by lipo-oligosaccharide (LOS) genotyping, amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE).

Methodology/Principal Findings

C. jejuni HS:23 was a predominant serotype among GBS patients (50%), and no specific serotype was significantly associated with GBS compared to enteritis. PCR screening showed that 38/49 (78%) of strains could be assigned to LOS classes A, B, C, or E. The class A locus (4/7 vs 3/39; p<0.01) was significantly associated in the GBS-related strains as compared to enteritis strains. All GBS/oculomotor related strains contained the class B locus; which was also detected in 46% of control strains. Overlapping clonal groups were defined by MLST, AFLP and PFGE for strains from patients with gastroenteritis and GBS. MLST defined 22 sequence types (STs) and 7 clonal complexes including 7 STs not previously identified (ST-3742, ST-3741, ST-3743, ST-3748, ST-3968, ST-3969 and ST-3970). C. jejuni HS:23 strains from patients with GBS or enteritis were clonal and all strains belonged to ST-403 complex. Concordance between LOS class B and ST-403 complex was revealed. AFLP defined 25 different types at 90% similarity. The predominant AFLP type AF-20 coincided with the C. jejuni HS:23 and ST-403 complex.

Conclusion/Significance

LOS genotyping, MLST, AFLP and PFGE helped to identify the HS:23 strains from GBS or enteritis patients as clonal. Overall, genotypes exclusive for enteritis or for GBS-related strains were not obtained although LOS class A was significantly associated with GBS strains. Particularly, the presence of a clonal and putative neuropathogenic C. jejuni HS:23 serotype may contribute to the high prevalence of C. jejuni related GBS in Bangladesh.     

Development of an HRM-based,safe and high-throughput genotyping system for two <Emphasis Type="Italic">low phytic acid</Emphasis> mutations in soybean

Two low phytic acid (lpa) mutants, Gm-lpa-ZC-2 (ZC-lpa) and Gm-lpa-TW-1 (TW-lpa), resulting from a G → A mutation in GmIPK1 and a 2-bp deletion in GmMIPS1, respectively, were previously developed to increase the nutritional value and environmental friendliness of soybean meal. Two functional CAPS markers were subsequently developed for genotyping plants carrying the two mutant genes; however, both are costly and time consuming and hence unsuitable for large-scale breeding use. In the present work, by integrating a quick DNA extraction protocol with an optimized high-resolution melting curve (HRM) analysis, we developed a fast and high-throughput genotyping system for the two mutations. In this system, (1) DNAs are extracted within half an hour using a protocol that only requires freezing and heating of leaf disks in two non-toxic solutions and can be directly used for PCR; (2) for genotyping, asymmetric PCRs with competitive primers are performed, and the samples are then discriminated and grouped through HRM analysis; and (3) all steps are performed in a 96-well plate, and hence adaptable to high-throughput genotyping. Although the system was developed for two lpa mutations, the general principle should be applicable to any other genes in soybean.     

ScreenClust: Advanced statistical software for supervised and unsupervised high resolution melting (HRM) analysis

Background: High resolution melting (HRM) is an emerging new method for interrogating and characterizing DNA samples. An important aspect of this technology is data analysis. Traditional HRM curves can be difficult to interpret and the method has been criticized for lack of statistical interrogation and arbitrary interpretation of results. Methods: Here we report the basic principles and first applications of a new statistical approach to HRM analysis addressing these concerns. Our method allows automated genotyping of unknown samples coupled with formal statistical information on the likelihood, if an unknown sample is of a known genotype (by discriminant analysis or “supervised learning”). It can also determine the assortment of alleles present (by cluster analysis or “unsupervised learning”) without a priori knowledge of the genotypes present. Conclusion: The new algorithms provide highly sensitive and specific auto-calling of genotypes from HRM data in both supervised an unsupervised analysis mode. The method is based on pure statistical interrogation of the data set with a high degree of standardization. The hypothesis-free unsupervised mode offers various possibilities for de novo HRM applications such as mutation discovery.     

Stacking of five favorable alleles for amylase content,fragrance and disease resistance into elite lines in rice (Oryza sativa) by using four HRM-based markers and a linked gel-based marker

Marker-assisted selection (MAS) for qualitative traits such as grain quality and resistance to certain diseases has proven to be highly effective. Multiple genes responsible for various quality components and disease resistances can be simultaneously stacked to boost the performance and to lengthen the commercial lifespan of high-yield varieties. Grain quality genes (fgr and Wx) and three disease resistance genes (Pita, Pik and Xa23) have been well characterized and used in MAS breeding. However, stacking all of them together into a single variety has not been reported. We reported here the stacking of the five genes into elite lines in rice. We achieved this through the development of functional markers from causal mutations at fgr, Wx and Pita, a gene-targeted marker at Pik and the use of a linked marker for Xa23. We employed and optimized the high-resolution melting (HRM) analysis method for use as the genotyping platform of fgr, Wx, Pik and Pita. By combining high-throughput DNA isolation, multiplex and nested-PCR methods, we showed that HRM could serve as cost-effective, highly automated, moderate-throughput and reliable non-gel genotyping platform for a small-scale MAS program.     

Host choice and human blood index of Anopheles pseudopunctipennis in a village of the Andean valleys of Bolivia

Background

Knockdown resistance (kdr) is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para -type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques.

Methods

Fluorescence-based assays based on 1) TaqMan probes and 2) high resolution melt (HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR), Heated Oligonucleotide Ligation Assay (HOLA), Sequence Specific Oligonucleotide Probe – Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost), and safety (requirement for hazardous chemicals).

Results

The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions) and the most specific (with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS-PCR, SSOP-ELISA, PCR Dot Blot and HOLA was fairly similar with a small number of failures and incorrect scores.

Conclusion

The results of blind genotyping trials of each assay indicate that where maximum sensitivity and specificity are required the TaqMan real-time assay is the preferred method. However, the cost of this assay, particularly in terms of initial capital outlay, is higher than that of some of the other methods. TaqMan assays using a PCR machine and fluorimeter are nearly as sensitive as real-time assays and provide a cost saving in capital expenditure. If price is a primary factor in assay choice then the AS-PCR, SSOP-ELISA, and HOLA are all reasonable alternatives with the SSOP-ELISA approach having the highest throughput.     

Dynamics of CRISPR Loci in Microevolutionary Process of Yersinia pestis Strains

The potential use of CRISPR loci genotyping to elucidate population dynamics and microevolution of 146 Yersinia pestis strains from different biovars and locations was investigated in this work. The majority of strains from the Orientalis biovar presented specific spacer arrays, allowing for the establishment of a CRISPR signature for their respective isolates. Twenty-one new spacers were found in the Y. pestis strains from plague foci in Brazil. Ninety-three (64%) strains were grouped in the G1 genotype, whereas the others were distributed in 35 genotypes. This study allowed observing a microevolutionary process in a group of Y. pestis isolated from Brazil. We also identified specific genotypes of Y. pestis that were important for the establishment of the bacteria in plague foci in Brazil. The data have provided supporting evidence for the diversity and dynamics of CRISPR loci present in the genome of Y. pestis strains from plague foci in Brazil.     

DNA barcode ITS2 coupled with high resolution melting (HRM) analysis for taxonomic identification of Sideritis species growing in Greece

Identification of genotypes in Sideritis is complicated owing to the morphological similarity and common occurrence of natural hybridisation within Sideritis species. Species- and genotype-specific DNA markers are very useful for plant identification, breeding and preservation programs. Herein, a real-time polymerase chain reaction (PCR) of ITS2 barcode region coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on seven Sideritis species growing in Greece. The HRM assay developed in this study is a rapid and straightforward method for the identification and discrimination of the investigated Sideritis species. This assay is simple compared to other genotyping methods as it does not require DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of Sideritis species.