High-throughput sex identification by melting curve analysis in blue-breasted quail and chicken

The objective was to develop a high-throughput method of identifying sex in both Coturnix chinensis and Gallus gallus, which would be useful for biomedical research and hatcheries. Because chromo-helicase-DNA binding protein (CHD)-based Griffiths P2/P8 primers do not produce polymerase chain reaction (PCR) products with distinguishable sex-specific curves in melting curve analysis (MCA), these primers are unsuitable for high throughput application in either species. Conserved regions were identified by basic local alignment search tool (BLAST) analyses of cloned CHD-Z and CHD-W genes of C. chinensis. Based on sequence alignment, a female-specific CHD-W primer (W-cot-F1) and a female/male (or CHD-W/CHD-Z)-common primer (ZW-cot-F1) were redesigned for use in combination with the Griffiths P2 primer for MCA-based PCR reaction. In C. chinensis and G. gallus, W-cot-F1/P2 and ZW-cot-F1/P2 had amplicon lengths of 315/318 and 114 base pairs and melting temperatures (Tm) of approximately 79.5 °C to 80 °C and approximately 78.5 °C to 79°C, respectively. Thus, MCA distinguished sex based on two distinct Tm peaks in females versus only one Tm peak in males. The MCA-based real-time PCR combined with the proposed primer redesign provided a high-throughput method of identifying sex in C. chinensis and G. gallus.     

High-Throughput Gender Identification of Penguin Species Using Melting Curve Analysis

Most species of penguins are sexual monomorphic and therefore it is difficult to visually identify their genders for monitoring population stability in terms of sex ratio analysis. In this study, we evaluated the suitability using melting curve analysis (MCA) for high-throughput gender identification of penguins. Preliminary test indicated that the Griffiths's P2/P8 primers were not suitable for MCA analysis. Based on sequence alignment of Chromo-Helicase-DNA binding protein (CHD)-W and CHD-Z genes from four species of penguins (Pygoscelis papua, Aptenodytes patagonicus, Spheniscus magellanicus, and Eudyptes chrysocome), we redesigned forward primers for the CHD-W/CHD-Z-common region (PGU-ZW2) and the CHD-W-specific region (PGU-W2) to be used in combination with the reverse Griffiths's P2 primer. When tested with P. papua samples, PCR using P2/PGU-ZW2 and P2/PGU-W2 primer sets generated two amplicons of 148- and 356-bp, respectively, which were easily resolved in 1.5% agarose gels. MCA analysis indicated the melting temperature (Tm) values for P2/PGU-ZW2 and P2/PGU-W2 amplicons of P. papua samples were 79.75°C–80.5°C and 81.0°C–81.5°C, respectively. Females displayed both ZW-common and W-specific Tm peaks, whereas male was positive only for ZW-common peak. Taken together, our redesigned primers coupled with MCA analysis allows precise high throughput gender identification for P. papua, and potentially for other penguin species such as A. patagonicus, S. magellanicus, and E. chrysocome as well.     

Validation of Spilornis cheela hoya TaqMan probes for potential gender identification of many Accipitridae species

The objective of this study was to test the hypothesis that genders of Accipitridae species, with the same or similar sequences to our previously proposed Spilornis cheela hoya (S. c. hoya) chromo-helicase-DNA binding protein (CHD)-W-specific and CHD-ZW-common TaqMan probes, can be successfully determined. Eight species of Accipitridae with known genders were collected. After PCR, TA cloning, sequencing, and alignment analyses, sequence length differences of Griffiths P2/P8 PCR amplicons between CHD-Z and CHD-W genes ranged from 2 to 19 bp for these Accipitridae species, and they were unsolved in 3% agarose gel. Using our previous proposed S. c. hoya TaqMan probes, the genders of Circaetus gallicus, completely homologous to the sequences for these CHD probes, were successfully identified. With one nucleotide difference to S. c. hoya CHD-W-specific probe, gender identification of Accipiter gularis, Accipiter soloensis, Accipiter trivirgatus, Accipiter virgatus, and Butastur indicus were validated. With two nucleotide differences in the CHD-W-specific probe and one nucleotide difference in the CHD-ZW-common probe, Pernis ptilorhyncus also performed well for gender identification. In conclusion, the S. c. hoyaCHD probes, coupled with the Griffiths P2/P8 primers, were validated to provide accurate and high-throughput gender identification for many Accipitridae species.     

Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

Background

Combination of CHD (chromo-helicase-DNA binding protein)-specific polymerase chain reaction (PCR) with electrophoresis (PCR/electrophoresis) is the most common avian molecular sexing technique but it is lab-intensive and gel-required. Gender determination often fails when the difference in length between the PCR products of CHD-Z and CHD-W genes is too short to be resolved.

Results

Here, we are the first to introduce a PCR-melting curve analysis (PCR/MCA) to identify the gender of birds by genomic DNA, which is gel-free, quick, and inexpensive. Spilornis cheela hoya (S. c. hoya) and Pycnonotus sinensis (P. sinensis) were used to illustrate this novel molecular sexing technique. The difference in the length of CHD genes in S. c. hoya and P. sinensis is 13-, and 52-bp, respectively. Using Griffiths' P2/P8 primers, molecular sexing failed both in PCR/electrophoresis of S. c. hoya and in PCR/MCA of S. c. hoya and P. sinensis. In contrast, we redesigned sex-specific primers to yield 185- and 112-bp PCR products for the CHD-Z and CHD-W genes of S. c. hoya, respectively, using PCR/MCA. Using this specific primer set, at least 13 samples of S. c. hoya were examined simultaneously and the Tm peaks of CHD-Z and CHD-W PCR products were distinguished.

Conclusion

In this study, we introduced a high-throughput avian molecular sexing technique and successfully applied it to two species. This new method holds a great potential for use in high throughput sexing of other avian species, as well.     

Rapid Identification of Medically Important Candida Isolates Using High Resolution Melting Analysis

An increasing trend in non albicans infections and various susceptibility patterns to antifungal agents implies a requirement for the quick and reliable identification of a number of medically important Candida species. Real-time PCR followed by high resolution melting analysis (HRMA) was developed, tested on 25 reference Candida collection strains and validated on an additional 143 clinical isolates in this study. All reference strains and clinical isolates inconclusive when using phenotypic methods and/or HRMA were analysed using ITS2 sequencing. Considering reference and clinical strains together, 23 out of 27 Candida species could be clearly distinguished by HRMA, while the remaining 4 species were grouped in 2 pairs, when applying the mean Tm ± 3 SD values, the shape of the derivative melting curve (dMelt curve) and, in some cases, the normalized and temperature—shifted difference plot against C. krusei. HRMA as a simple, rapid and inexpensive tool was shown to be useful in identifying a wide spectrum of clinically important Candida species. It may complement the current clinical diagnostic approach based on commercially available biochemical kits.     

Quantitative Detection and Differentiation of Free-Living Amoeba Species Using SYBR Green–Based Real-Time PCR Melting Curve Analysis

Real-time polymerase chain reaction melting curve analysis (MCA) allows differentiation of several free-living amoebae species. Distinctive characteristics were found for Naegleria fowleri, N. lovaniensis, N. australiensis, N. gruberi, Hartmanella vermiformis, and Willaertia magna. Species specificity of the amplicons was confirmed using agarose gel electrophoresis and sequence-based approaches. Amplification efficiency ranged from 91% to 98%, indicating the quantitative potential of the assay. This MCA approach can be used for quantitative detection of free-living amoebae after cultivation but also as a culture-independent detection method.     

Design of molecular markers for identification of species of the genus Chironomus (Diptera,Chironomidae)

Accurate identification and differentiation of species of the genus Chironomus based on their morphological features is a difficult problem. Unambiguous species identification by means of molecular markers is possible at any stage of the life cycle. Polymerase chain reaction (PCR) with species-specific primers was used to develop molecular markers (amplicons) for identification of Chironomus piger, Ch. dorsalis, and Ch. pseudothummi. Nucleotide sequences of the internal transcribed spacer region (ITS) of the locus coding for ribosomal RNA were used to design species-specific primers for these target species. Each of the species-specific primer pairs yielded species-specific amplicons (molecular markers) only with the DNA of target species: Ch. piger, Ch. dorsalis, and Ch. pseudothummi. Test PCRs with the DNA of eighteen Chironomus species confirmed the specificity of the primers obtained. The molecular markers produced in PCR with the designed species-specific primers permit reliable identification of Ch. piger, Ch. dorsalis, and Ch. pseudothummi and their differentiation from other species of the genus Chironomus.     

Evaluation of real-time PCR targeting hbb gene for Borrelia species identification

Several molecular methods have been employed for Borrelia species identification. Newly developed technology, real-time polymerase chain reaction (RT-PCR), combines simultaneous amplification, detection and differentiation of strains in one PCR run. The aim of the study was to perform and evaluate RT-PCR for Borreliaburgdorferi sensu lato species identification. Borrelia species identification was accomplished on 374 Borrelia strains using two approaches: 1.) MluI restriction of entire borrelial chromosome (MluI-large restriction fragment patterns, LRFP), and 2.) RT-PCR targeting hbb gene and specific melting temperature (Tm) detection. The results of the two molecular methods were compared. With MluI-RFLP we were able to differentiate all Borrelia species and their subtypes within particular species. RT-PCR based on Tm determination identified unique strains within the species Borreliaafzelii (Tm 66.11 °C), B. burgdorferi sensu stricto (Tm 68.18 °C), Borreliaspielmanii (Tm 59.45 °C) and Borreliavalaisiana (Tm 59.62 °C). We were not able to distinguish the last two species that shared almost identical Tm. The large majority of Borreliagarinii strains shared Tm 51.42 °C, while subtype Mlg4 was characterized by Tm 56.87 °C. Strains of Borrelialusitaniae species also were heterogeneous; human isolate had Tm 63.47 °C while two tick isolates shared Tm 61.77 °C. Differences inside hbb gene enabled differentiation of the majority of Borrelia species, and revealed two clusters within B. garinii and B. lusitaniae species, respectively, but it was not possible to distinguish B. spielmanii form B. valaisiana. The major advantage of RT-PCR was that it was easy to perform and that the results were obtained within a few hours.     

Design and Validation of Four New Primers for Next-Generation Sequencing To Target the 18S rRNA Genes of Gastrointestinal Ciliate Protozoa

Four new primers and one published primer were used to PCR amplify hypervariable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST database (NCBI) and MOTHUR version 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F and GIC758R (amplicon of 482 bases), gave the best representation of diversity using BLAST classification, and the set amplified Entodinium simplex and Ostracodinium spp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F and GIC1578R (amplicon of 498 bases), had similar BLAST results and a slightly higher percentage of sequences that were identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in nonruminants. The amplicons created by primer set 1 were indistinguishable for certain species within the genera Bandia, Blepharocorys, Polycosta, and Tetratoxum and between Hemiprorodon gymnoprosthium and Prorodonopsis coli, none of which are normally found in the rumen.     

Rapid and Accurate Identification of Mycobacterium tuberculosis Complex and Common Non-Tuberculous Mycobacteria by Multiplex Real-Time PCR Targeting Different Housekeeping Genes

Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T _m) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100?% for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100?%, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.     

Allele-specific extension allows base-pair neutral homozygotes to be discriminated by high-resolution melting of small amplicons

Not all single-nucleotide polymorphisms (SNPs) can be determined using high-resolution melting (HRM) of small amplicons, especially class 3 and 4 SNPs. This is due mainly to the small shift in the melting temperature (Tm) between two types of homozygote. Choosing rs1869458 (a class 4 SNP) as a sample, we developed a modified small amplicon HRM assay. An allele-specific extension (ASE) primer, which ended at an SNP site and matched only one of the alleles, was added to the reaction as well as additional thermal steps for ASE. Following asymmetric polymerase chain reaction and melting curve analysis, heterozygotes were easily identified. Two types of homozygote were also distinguishable, indicating that extension primers 11 to 13 bases in length worked efficiently in an allele-specific way. Modification of the limiting amplification primer with locked nucleic acid increased the Tm difference between extension and amplification peaks and facilitated subsequent genotyping. In addition, 194 human genomic DNA samples were genotyped with the developed assay and by direct sequencing, with the different methods providing identical genotyping results. In conclusion, ASE-HRM is a simple, inexpensive, closed-tube genotyping method that can be used to examine all types of SNP.     

Rapid differentiation of bacterial species by high resolution melting curve analysis

Molecular based differentiation of various bacterial species is important in phylogenetic studies, diagnostics and epidemiological surveillance, particularly where unusual phenotype makes the classical phenotypic identification of bacteria difficult. Molecular approach based on the sequence of 16S ribosomal RNA gene analysis can achieve fast and reliable identification of bacteria. High resolution melting (HRM) curve analysis has been developed as an attractive novel technique for DNA sequence discrimination but it’s application for bacteria differentiation has not been well studied yet. We have developed HRM assay for differentiation of sixteen pathogenic or opportunistic bacterial species. Amplified partial 16S ribosomal RNA gene region between 968 and 1401 positions (E. coli reference numbering) was subsequently used in high resolution melting curve analysis of PCR products for bacterial species differentiation. Sixteen bacterial species were simultaneously discerned by difference plot of normalized and temperatures shifted melting curves, without need for spiking of DNA, hetero-duplexing experiments or application of several primer pairs. High resolution melting curve analysis of duplex DNA is simple, fast and reliable tool for bacterial species differentiation and may efficiently complement phenotypic identification of bacteria.     

PCR-based rapid diagnostic tools for the authentication of medicinal mistletoe species

BackgroundTaxilli Herba (TH) and Visci Herba (VH), defined as the leaves and branches of the mistletoe species Taxillus chinensis and Viscum coloratum, respectively, are popular herbal medicines in East Asia. However, commercial TH and VH products are frequently adulterated with related inauthentic mistletoe species, posing efficacy and safety concerns. Accurate species identification of herbal medicinal products is a prerequisite for quality control, but traditional morphological identification methods are hampered by difficulties in discriminating among closely related species and in identifying the source materials in processed products.PurposeThis study aimed to develop sequence-characterized amplified region (SCAR) markers and a multiplex-SCAR assay for rapid and accurate identification of authentic TH and VH.MethodsThe matK region was sequenced in a total of 20 samples from five mistletoe species, namely T. chinensis and V. coloratum, and three species often found in adulterated herbal medicines, T. sutchuenensis, V. articulatum, and Macrosolen tricolor. Species-specific nucleotide polymorphisms were identified and short regions (21–22 bp) containing at least two species-specific nucleotides close to the 3ʹ end were incorporated into SCAR primers that produced uniquely sized PCR amplicons for each species. The five SCAR primer sets were also combined into a multiplex-SCAR assay.ResultsThe SCAR primers successfully generated amplicons of the expected size for each target species even with low-DNA templates or with templates containing DNA from multiple samples. No amplification was observed in non-target species. The SCAR markers and the multiplex-SCAR assay successfully identified commercial TH and VH products that were counterfeit or adulterated in both dried and processed products.ConclusionThis is the first report to illustrate discrimination of genuine medicinal mistletoe species with DNA-based marker assays, enabling rapid and accurate species identification. The SCAR assays developed in this study will facilitate the standardization of commercial mistletoe products.     

Rapid identification of chloroplast haplotypes using High Resolution Melting analysis

We have evaluated High Resolution Melting (HRM) analysis as a method for one‐step haplotype identification in phylogeographic analysis. Using two adjoined internal amplicons (c. 360 and 390 bp) at the chloroplast rps16 intron (c. 750 bp) we applied HRM to identify haplotypes in 21 populations of two European arctic‐alpine herb species Arenaria ciliata and Arenaria norvegica (Caryophyllaceae). From 446 accessions studied, 20 composite rps16 haplotypes were identified by the melting‐curve protocol, 18 of which could be identified uniquely. In a comparative sensitivity analysis with in silico PCR‐RFLP, only seven of these 20 haplotypes could be identified uniquely. Observed in vitro experimental HRM profiles were corroborated by in silico HRM analysis generated on uMelt^SM. In silico mutation analysis carried out on a 360 bp wild‐type rps16I amplicon determined that the expected rate of missed single‐nucleotide polymorphisms (SNP) detection in vitro was similar to existing evaluations of HRM sensitivity, with transversion SNPs being more likely to go undetected compared to transition SNPs. In vitro HRM successfully discriminated between all amplicon templates differing by two or more base changes (352 cases) and between 11 pairs of amplicons where the only difference was a single transition or transversion SNP. Only one pairwise comparison yielded no discernable HRM curve difference between haplotypes, and these samples differed by one transversion (C/G) SNP. HRM analysis represents an untapped resource in phylogeographic analysis, and with appropriate primer design any polymorphic locus is potentially amenable to this single‐reaction method for haplotype identification.     

Chromosomal evolution of South American Columbiformes (Aves)

Karyotypes are compared of 14 species of Brazilian Columbiformes (family Columbidae): Claravis pretiosa (2n=74), Columba cayennensis (2n=76), Columba picazuro (2n=76), Columba speciosa (2n=76), Columbina minuta (2n=76), Columbina passerina (2n=76), Columbina picui (2n=76), Columbina talpacoti (2n=76), Geotrygon montana (2n=86), Leptotila rufaxilla (2n=76), Leptotila verreauxi (2n=78), Scardafella squammata (2n=78), Uropelia campestris (2n=68) and Zenaida auriculata (2n=76). The macrochromosomes of each species were analysed by conventional Giemsa staining, cytobiometrically and with G-and C-banding.All species studied are characterized by typical bird karyotypes with a few pairs of macrochromosomes and many microchromosomes.The morphology and relative length of the Z chromosome are nearly the same in all species, but the W chromosome shows variation. The G-band patterns of the first pair in Columbiformes show a large positive band distally in the long arm, common to all species of the order. The constitutive heterochromatin is restricted to the centromeres of the macro- and microchromosomes. The W is the most heterochromatic chromosome in all species studied.Studies of relative lengths, arm ratios and G- and C-banding patterns showed that in Columbiformes pairs 3, 4 and 5 are the most stable. The types of rearrangements distinguishing between species vary among the genera: pericentric inversions in Columba; fusions and translocations in Uropelia; centric fissions in Geotrygon; fusions, translocations, para and pericentric inversions in Columbina, Leptotila, Zenaida and Scardafella.On the basis of the karyological findings the phylogenetic relationships of the Brazilian Columbiformes are discussed.This work was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (Plano Integrado de Genética-Processo No. 22.1375/77 and 40.0499/80).     

Rapid, Sensitive, and Discriminating Identification of Naegleria spp. by Real-Time PCR and Melting-Curve Analysis

The free-living amoeboflagellate genus Naegleria includes one pathogenic and two potentially pathogenic species (Naegleria fowleri, Naegleria italica, and Naegleria australiensis) plus numerous benign organisms. Monitoring of bathing water, water supplies, and cooling systems for these pathogens requires a timely and reliable method for identification, but current DNA sequence-based methods identify only N. fowleri or require full sequencing to identify other species in the genus. A novel closed-tube method for distinguishing thermophilic Naegleria species is presented, using a single primer set and the DNA intercalating dye SYTO9 for real-time PCR and melting-curve analysis of the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1, ITS2). Collection of DNA melting data at close temperature intervals produces highly informative melting curves with one or more recognizable melting peaks, readily distinguished for seven Naegleria species and the related Willaertia magna. Advantages over other methods used to identify these organisms include its comprehensiveness (encompassing all species tested to date), simplicity (no electrophoresis required to verify the product), and sensitivity (unambiguous identification from DNA equivalent to one cell). This approach should be applicable to a wide range of microorganisms of medical importance.     

Identification of fibrillin-1 gene mutations in Marfan syndrome by high-resolution melting analysis

Marfan syndrome has been associated with approximately 562 mutations in the fibrillin-1 (FBN1) gene. Mutation scanning of the FBN1 gene with DNA direct sequencing is time-consuming and expensive because of its large size. This study analyzed the diagnostic value of high-resolution melting analysis as an alternative method for scanning of the FBN1 gene. A total of 75 polymerase chain reaction (PCR) amplicons (179-301 bp, average 256 bp) that covered the complete coding regions and splicing sites were evaluated on the 96-well LightCycler system. Melting curves were analyzed as fluorescence derivative plots (−dF/dT vs. temperature). To determine the sensitivity of this method, a total of 82 samples from patients with Marfan syndrome and 50 unaffected individuals were analyzed. All mutations reported in this study had been confirmed previously by direct sequencing analysis. Melting analysis identified 48 heterozygous variants. The variant c.3093 G>T (exon 25) was incorrectly identified by melting curve analysis. The sensitivity of the technique in this sample was 98.78% (81/82). This study demonstrated that high-resolution melting analysis is a reliable gene scanning method with greater speed than DNA sequencing. Our results support the use of this technology as an alternative method for the diagnosis of Marfan syndrome as well as its suitability for high-throughput mutation scanning of other large genes.     

Species-specific ITS primers for the identification of Picoa juniperi and Picoa lefebvrei and using nested-PCR for detection of P. juniperi in planta

Desert truffles, hypogeous Pezizales (Ascomycota), are difficult to identify due to evolutionary convergence of morphological characters among taxa that share a similar habitat and mode of spore dispersal. Also, during their symbiotic phase, these are barely distinguishable morphologically, and molecular probes are needed for their identification. We have developed a PCR-based method for the identification of Picoa juniperi and Picoa lefebvrei based on internal transcribed spacers of rDNA. Two PCR primers specific for P. lefebvrei (FLE/RLE) and two specific for P. juniperi (FJU/RJU) were designed. A collection of samples from different geographical areas representing diversity of these species were examined for unique regions of internal transcribed spacers 1, 2 and 5.8S gene of rDNA (ITS) compared to other closely related species. Annealing temperatures and extension times were optimized for each set of primers for maximum specificity and efficiency. They proved to be efficient to specifically detect the presence of P. juniperi and P. lefebvrei by PCR and neither set amplified purified DNA from other truffle species as well as some ascomycetous fungi. The partial small subunit of ribosomal DNA genes of P. juniperi were amplified with the genomic DNA extracted from Helianthemum ledifolium var. ledifolium roots by nested polymerase chain reaction (PCR) using the universal fungal primer pair ITS1/ITS4 and specific primer pair FTC/RTC, which was designed based on internal transcribed spacer 1, 2 and 5.8S gene of rDNA sequences of P juniperi. The nested-PCR was sensitive enough to re-amplify the direct-PCR product, resulting in a DNA fragment of 426 bp. The efficacy of nested-PCR showed that it could re-amplify the direct-PCR product and detect 200 fg genomic DNA.     

SMM-system: A mining tool to identify specific markers in Salmonella enterica

This report presents SMM-system, a software package that implements various personalized pre- and post-BLASTN tasks for mining specific markers of microbial pathogens. The main functionalities of SMM-system are summarized as follows: (i) converting multi-FASTA file, (ii) cutting interesting genomic sequence, (iii) automatic high-throughput BLASTN searches, and (iv) screening target sequences. The utility of SMM-system was demonstrated by using it to identify 214 Salmonella enterica-specific protein-coding sequences (CDSs). Eighteen primer pairs were designed based on eighteen S. enterica-specific CDSs, respectively. Seven of these primer pairs were validated with PCR assay, which showed 100% inclusivity for the 101 S. enterica genomes and 100% exclusivity of 30 non-S. enterica genomes. Three specific primer pairs were chosen to develop a multiplex PCR assay, which generated specific amplicons with a size of 180 bp (SC1286), 238 bp (SC1598) and 405 bp (SC4361), respectively. This study demonstrates that SMM-system is a high-throughput specific marker generation tool that can be used to identify genus-, species-, serogroup- and even serovar-specific DNA sequences of microbial pathogens, which has a potential to be applied in food industries, diagnostics and taxonomic studies. SMM-system is freely available and can be downloaded from http://foodsafety.sjtu.edu.cn/SMM-system.html.     

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.