Development of amplified fragment length polymorphism (AFLP)‐derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot

Aims

The objective of this work was to design an amplified fragment length polymorphism (AFLP)‐derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot (PBRR) in plant material and soil.

Methods and Results

Specific primers for the detection of the pathogen were designed based on an amplified region using AFLPs. The banding patterns by AFLPs showed that isolates from diseased roots were clearly distinguishable from others members of the F. solani species complex. Many bands were specific to F. solani PBRR, one of these fragments was selected and sequenced. Sequence obtained was used to develop specific PCR primers for the identification of pathogen in pure culture and in plant material and soil. Primer pair FS1/FS2 amplified a single DNA product of 175 bp. Other fungal isolates occurring in soil, included F. solani non‐PBRR, were not detected by these specific primers. The assay was effective for the detection of pathogen from diseased root and infected soils.

Conclusions

The designed primers for F. solani causing PBRR can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen.

Significance and Impact of the Study

These diagnostic PCR primers will aid the detection of F. solani causing PBRR in diseased root and natural infected soils. The method developed could be a helpful tool for epidemiological studies and to avoid the spread of this serious disease in new areas.     

PathogenMIPer: a tool for the design of molecular inversion probes to detect multiple pathogens

Background  

Here we describe PathogenMIPer, a software program for designing molecular inversion probe (MIP) oligonucleotides for use in pathogen identification and detection. The software designs unique and specific oligonucleotide probes targeting microbial or other genomes. The tool tailors all probe sequence components (including target-specific sequences, barcode sequences, universal primers and restriction sites) and combines these components into ready-to-order probes for use in a MIP assay. The system can harness the genetic variability available in an entire genome in designing specific probes for the detection of multiple co-infections in a single tube using a MIP assay.     

Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor?

Background  

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor^? software, aimed at simultaneous detection of mutations in three homoeologous genes.     

Delineating the structural,functional and evolutionary relationships of sucrose phosphate synthase gene family II in wheat and related grasses

Background  

Sucrose phosphate synthase (SPS) is an important component of the plant sucrose biosynthesis pathway. In the monocotyledonous Poaceae, five SPS genes have been identified. Here we present a detailed analysis of the wheat SPSII family in wheat. A set of homoeologue-specific primers was developed in order to permit both the detection of sequence variation, and the dissection of the individual contribution of each homoeologue to the global expression of SPSII.     

Loop‐mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp

Aims

Enterocytozoon hepatopenaei is an emerging microsporidian parasite that has been linked to recent losses caused by white faeces syndrome (WFS) in cultivated giant or black tiger shrimp Penaeus (Penaeus) monodon and whiteleg shrimp Penaeus (Litopenaeus) vannamei in Asia. To more accurately assess its impact on shrimp production and to determine reservoir carriers for control measures, our objective was to establish a loop‐mediated isothermal amplification (LAMP) assay combined with colorimetric nanogold (AuNP) for rapid, sensitive and inexpensive detection of this parasite.

Methods and Results

A set of six specific primers was designed to successfully detect the SSU rRNA gene of E. hepatopenaei by a LAMP reaction of 45 min at 65°C combined with visual detection of the amplification product via hybridization at 65°C for 5 min with a ssDNA‐labelled nanogold probe, followed by salt‐induced AuNP aggregation (total assay time, approximately 50 min). This method gave similar results to LAMP followed by electrophoresis or spectrophotometric detection, and it was more sensitive (0·02 fg total DNA) than a conventional nested PCR (0·2 fg total DNA). The new method gave negative results with shrimp DNA templates extracted from diseased shrimp containing other pathogens, indicating that the LAMP‐AuNP assay was specific for E. hepatopenaei.

Conclusions

Without sacrificing sensitivity or specificity, the new LAMP‐AuNP assay significantly reduced the time, ease and cost for molecular detection of E. hepatopenaei in shrimp.

Significance and Impact of the study

The new method employs simple, inexpensive equipment and involves simple steps making it applicable for small field laboratories. Wider application of the method to screen broodstock before use in a hatchery, to screen postlarvae before stocking shrimp ponds, to test for natural carriers and to monitor shrimp in rearing ponds would help to assess and reduce the negative impact of this parasite in shrimp farming.     

Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies

Background  

In protein engineering, site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions. In a widely used method, mutations are generated by PCR using a pair of oligonucleotide primers designed with mismatching nucleotides at the center of the primers. In this method, primer-primer annealing may prevent cloning of mutant cDNAs. To circumvent this problem we developed an alternative procedure that does not use forward-reverse primer pair in the same reaction.     

Selection strategy and the design of hybrid oligonucleotide primers for RACE-PCR: cloning a family of toxin-like sequences from <Emphasis Type="Italic">Agelena orientalis</Emphasis>

Background  

the use of specific but partially degenerate primers for nucleic acid hybridisations and PCRs amplification of known or unknown gene families was first reported well over a decade ago and the technique has been used widely since then.     

False positive PCR detection of <Emphasis Type="Italic">Tropheryma whipplei</Emphasis> in the saliva of healthy people

Background  

Tropheryma whipplei, the agent of Whipple's disease (WD), has been recently isolated and the genomes of two isolates have been fully sequenced. Previous diagnosis tools for the diagnosis of the disease used sequence analysis of the 16S rRNA gene. Using this target gene, the high percentage of detection of the bacterium in saliva of healthy people was in contrast to the negative results obtained with specific target genes. The aim of our study was to compare previously published primers targeting the 16S rRNA gene to real-time PCR with Taqman* probes targeting specific repeat genes only found in the genome of T. whipplei in a series of 57 saliva from healthy people.     

Rapid identification of bacterial pathogens using a PCR- and microarray-based assay

Background  

During the course of a bacterial infection, the rapid identification of the causative agent(s) is necessary for the determination of effective treatment options. We have developed a method based on a modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 12 bacterial pathogens at the species level. The broad-range PCR primer mixture was designed using conserved regions of the bacterial topoisomerase genes gyrB and parE. The primer design allowed the use of a novel DNA amplification method, which produced labeled, single-stranded DNA suitable for microarray hybridization. The probes on the microarray were designed from the alignments of species- or genus-specific variable regions of the gyrB and parE genes flanked by the primers. We included mecA-specific primers and probes in the same assay to indicate the presence of methicillin resistance in the bacterial species. The feasibility of this assay in routine diagnostic testing was evaluated using 146 blood culture positive and 40 blood culture negative samples.     

Identification of ochratoxin A producing Aspergillus carbonarius and A. niger clade isolated from grapes using the loop‐mediated isothermal amplification (LAMP) reaction

Aims

To develop two assays based on the loop‐mediated isothermal amplification (LAMP) of DNA for the quick and specific identification of Aspergillus carbonarius and ochratoxigenic strains of the Aspergillus niger clade isolated from grapes.

Methods and Results

Two sets of primers were designed based on the polyketide synthase genes involved or putatively involved in ochratoxin A (OTA) biosynthesis in A. carbonarius and A. niger clade. Hydroxynaphthol blue was used as indirect method to indicate DNA amplification. The limit of detection of both assays was comparable to that of a PCR reaction. Specificities of the reactions were tested using DNA from different black aspergilli isolated from grapes. The two LAMP assays were then used to identify A. carbonarius and ochratoxigenic A. niger and A. awamori grown in pure cultures without a prior DNA extraction.

Conclusions

The two LAMP assays permitted to quickly and specifically identify DNA from OTA‐producing black aspergilli, as well as isolates grown in pure culture.

Significance and Impact of the Study

Monitoring vineyards for the presence of OTA‐producing strains is part of the measures to minimize the occurrence of OTA in grape products. The two LAMP assays developed here could be potentially used to speed the screening process of vineyards for the presence of OTA‐producing black aspergilli.     

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V‐C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA‐A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl₂, Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low‐cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V‐C.

Significance and Impact of the Study

Incidence of phytoplasma diseases is increasing worldwide and particularly in the tropical and subtropical world. Co‐infection of phytoplasma and virus(s) is also common. Therefore, use of single primer PCR in detecting these pathogens would require more time and effort, whereas multiplex PCR involving several pairs of primers saves time and reduces cost. In this study, we have developed a multiplex nested PCR assay that provides more sensitive and specific detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma in white jute simultaneously. It is the first report of simultaneous detection of CoGMV and a phytoplasma in Corchorus capsularis by multiplex nested PCR.     

Hepatitis C virus whole genome position weight matrix and robust primer design

Background  

The high degree of sequence heterogeneity found in Hepatitis C virus (HCV) isolates, makes robust nucleic acid-based assays difficult to generate. Polymerase chain reaction based techniques, require efficient and specific sequence recognition. Generation of robust primers capable of recognizing a wide range of isolates is a difficult task.     

Development of a sensitive molecular detection assay for mango malformation disease caused by <Emphasis Type="Italic">Fusarium mangiferae</Emphasis>

Objectives

To develop a sensitive and specific molecular assay for detection of mango malformation disease (MMD), which is caused primarily by Fusarium mangiferae.

Results

We screened 100 ISSR primers and identified one (UBC888) that directed the stable amplification of a specific gene fragment of 479 bp (GenBank accession number KJ526382). Based on the DNA sequence of this fragment, a pair of SCAR primers (W342 and W1772) were designed to amplify another gene fragment of 1376 bp (GenBank accession number KJ526383), demonstrating the successful conversion of an ISSR marker to a SCAR marker. An effective and simple detection assay for MMD was established based on this pair of PCR primers, with a high level of specificity and sensitivity to the DNA of F. mangiferae and other species of Fusarium both in vitro and in vivo. It can detect as little as 10 pg fungal DNA from the DNA of mango’s tissues.

Conclusions

Our assay provides a practical method for the early diagnosis so that proper prevention of the mango malformation disease can be developed.

     

ConservedPrimers 2.0: A high-throughput pipeline for comparative genome referenced intron-flanking PCR primer design and its application in wheat SNP discovery

Background  

In some genomic applications it is necessary to design large numbers of PCR primers in exons flanking one or several introns on the basis of orthologous gene sequences in related species. The primer pairs designed by this target gene approach are called "intron-flanking primers" or because they are located in exonic sequences which are usually conserved between related species, "conserved primers". They are useful for large-scale single nucleotide polymorphism (SNP) discovery and marker development, especially in species, such as wheat, for which a large number of ESTs are available but for which genome sequences and intron/exon boundaries are not available. To date, no suitable high-throughput tool is available for this purpose.     

Unsupervised reduction of random noise in complex data by a row-specific,sorted principal component-guided method

Background  

Large biological data sets, such as expression profiles, benefit from reduction of random noise. Principal component (PC) analysis has been used for this purpose, but it tends to remove small features as well as random noise.     

GONOME: measuring correlations between GO terms and genomic positions

Background

The design of oligonucleotides and PCR primers for studying large genomes is complicated by the redundancy of sequences. The eukaryotic genomes are particularly difficult to study due to abundant repeats. The speed of most existing primer evaluation programs is not sufficient for large-scale experiments.

Results

In order to improve the efficiency and success rate of automatic primer/oligo design, we created a novel method which allows rapid masking of repeats in large sequence files, for example in eukaryotic genomes. It also allows the detection of all alternative binding sites of PCR primers and the prediction of PCR products. The new method was implemented in a collection of efficient programs, the GENOMEMASKER package. The performance of the programs was compared to other similar programs. We also modified the PRIMER3 program, to be able to design primers from lowercase-masked sequences.

Conclusion

The GENOMEMASKER package is able to mask the entire human genome for non-unique primers within 6 hours and find locations of all binding sites for 10 000 designed primer pairs within 10 minutes. Additionally, it predicts all alternative PCR products from large genomes for given primer pairs.