Detection Of Single Nucleotide Polymorphisms Research Articles

Single nucleotide polymorphisms of interleukin-35 subunit genes predict host susceptibility to chronic hepatitis B virus infection among Iraqi patients

Chronic hepatitis B virus (HBV) infection represents a complication of a major concern in public health. Interleukin-35 (IL-35) has been suggested to play a role in maintaining the viral persistence. It consists of two subunits (IL-12p35 and EBI3) that are coded by two genes located on different chromosomes; IL12A (3q25.33) and EBI3 (19p13.3), respectively. Single nucleotide polymorphisms (SNPs) of both genes have been associated with susceptibility to different diseases. Therefore, this study examined two intronic SNPs of IL12A (rs582054 and rs583911 in intron 2) and EBI3 (rs428253 and rs7254021 in intron 1) genes in 80 chronic HBV patients and 96 control with the aim to understand their susceptibility role in the disease. The method of SNP detection was allele-specific PCR using specific primers. Frequencies of rs582054 A allele and AT genotype were significantly increased in patients compared to control. C allele and CC genotype frequencies of rs428253 SNP were also significantly increased in patients. For rs583911 and rs7254021 SNPs, allele and genotype frequencies demonstrated no significant variation between patients and control. Haplotype analysis revealed that A-A haplotype of IL12A SNPs (rs582054 and rs583911) was associated with a significantly increased HBV risk. In contrary, G-C haplotype of EBI3 SNPs (rs428253 and rs7254021) was associated with a significant decreased risk. In conclusion, IL-35 subunit gene SNPs (IL12A rs582054 and EBI3 rs428253) are associated with susceptibility to develop chronic HBV infection among Iraqi patients.

Happy Birthday to Bernd Giese

Happy Birthday to Bernd Giese

RLP/K enrichment sequencing; a novel method to identify receptor-like protein (RLP) and receptor-like kinase (RLK) genes.

Summary The identification of immune receptors in crop plants is time‐consuming but important for disease control. Previously, resistance gene enrichment sequencing (RenSeq) was developed to accelerate mapping of nucleotide‐binding domain and leucine‐rich repeat containing (NLR) genes. However, resistances mediated by pattern recognition receptors (PRRs) remain less utilized.Here, our pipeline shows accelerated mapping of PRRs. Effectoromics leads to precise identification of plants with target PRRs, and subsequent RLP/K enrichment sequencing (RLP/KSeq) leads to detection of informative single nucleotide polymorphisms that are linked to the trait.Using Phytophthora infestans as a model, we identified Solanum microdontum plants that recognize the apoplastic effectors INF1 or SCR74. RLP/KSeq in a segregating Solanum population confirmed the localization of the INF1 receptor on chromosome 12, and led to the rapid mapping of the response to SCR74 to chromosome 9. By using markers obtained from RLP/KSeq in conjunction with additional markers, we fine‐mapped the SCR74 receptor to a 43‐kbp G‐LecRK locus.Our findings show that RLP/KSeq enables rapid mapping of PRRs and is especially beneficial for crop plants with large and complex genomes. This work will enable the elucidation and characterization of the nonNLR plant immune receptors and ultimately facilitate informed resistance breeding.

Highly selective detection of single nucleotide polymorphism (SNP) using a dumbbell DNA probe with a gap-filling approach

Single nucleotide polymorphism (SNP) is the most common type of genetic sequence variations and known to associate with the development of various diseases including cancer. Accurate detection of SNP is often necessary to determine the susceptibility of patients to diseases as well as their response to the medical treatments. Molecular inversion probe (MIP) has been widely utilized as an alternative method to detect the SNP as compared to the conventional PCR/genome sequencing method. However, base-pairing based detection of SNP fundamentally lacks target specificity toward the single mismatch of bases and often fails to meet the accuracy requirement for SNP detection. In this study, we report the development of new SNP detection method using a dumbbell-probe with a gap-filling approach. Two separate DNA strands are designed to self-assemble to form a dumbbell DNA probe for the selective detection of EGFR 21 point mutation. Unlike the direct binding of MIP to the target sequence, our system has an additional process for the enhancement of sequence specificity through a base matched gap-filling. In the presence of target sequences, the dumbbell-probe undergoes base-pair hybridization to the target sequence followed by sequence specific gap-filling and ligation in single-step. The detection of EGFR 21 point mutation is achieved with a highly specific and selective manner. This simple and robust detection method offers promising future potential in the diagnosis of various genetic mutations.

Detection of low-frequency resistance-mediating SNPs in next-generation sequencing data of Mycobacterium tuberculosis complex strains with binoSNP

Accurate drug resistance detection is key for guiding effective tuberculosis treatment. While genotypic resistance can be rapidly detected by molecular methods, their application is challenged by mixed mycobacterial populations comprising both susceptible and resistant cells (heteroresistance). For this, next-generation sequencing (NGS) based approaches promise the determination of variants even at low frequencies. However, accurate methods for a valid detection of low-frequency variants in NGS data are currently lacking. To tackle this problem, we developed the variant detection tool binoSNP which allows the determination of low-frequency single nucleotide polymorphisms (SNPs) in NGS datasets from Mycobacterium tuberculosis complex (MTBC) strains. By taking a reference-mapped file as input, binoSNP evaluates each genomic position of interest using a binomial test procedure. binoSNP was validated using in-silico, in-vitro, and serial patient isolates datasets comprising varying genomic coverage depths (100-500×) and SNP allele frequencies (1-30%). Overall, the detection limit for low-frequency SNPs depends on the combination of coverage depth and allele frequency of the resistance-associated mutation. binoSNP allows for valid detection of resistance associated SNPs at a 1% frequency with a coverage ≥400×. In conclusion, binoSNP provides a valid approach to detect low-frequency resistance-mediating SNPs in NGS data from clinical MTBC strains. It can be implemented in automated, end-user friendly analysis tools for NGS data and is a step forward towards individualized TB therapy.

Single nucleotide polymorphism-based analysis of the genetic structure of Liangshan pig population.

ObjectiveTo conserve and utilize the genetic resources of a traditional Chinese indigenous pig breed, Liangshan pig, we assessed the genetic diversity, genetic structure, and genetic distance in this study.MethodsWe used 50K single nucleotide polymorphism (SNP) chip for SNP detection of 139 individuals in the Liangshan Pig Conservation Farm.ResultsThe genetically closed conserved population consisted of five overlapping generations, and the total effective content of the population (Ne) was 15. The whole population was divided into five boar families and one non-boar family. Among them, the effective size of each generation subpopulation continuously decreased. However, the proportion of polymorphic markers (PN) first decreased and then increased. The average genetic distance of these 139 Liangshan pigs was 0.2823±0.0259, and the average genetic distance of the 14 boars was 0.2723±0.0384. Thus, it can be deduced that the genetic distance changed from generation to generation. In the conserved population, 983 runs of homozygosity (ROH) were detected, and the majority of ROH (80%) were within 100 Mb. The inbreeding coefficient calculated based on ROH showed an average value of 0.026 for the whole population. In addition, the inbreeding coefficient of each generation subpopulation initially increased and then decreased. In the pedigree of the whole conserved population, the error rate of paternal information was more than 11.35% while the maternal information was more than 2.13%.ConclusionThis molecular study of the population genetic structure of Liangshan pig showed loss of genetic diversity during the closed cross-generation reproduction process. It is necessary to improve the mating plan or introduce new outside blood to ensure long-term preservation of Liangshan pig.

High frequency of benzimidazole resistance alleles in trichostrongyloids from Austrian sheep flocks in an alpine transhumance management system

BackgroundInfections of small ruminants with trichostrongyloid nematodes often result in reduced productivity and may be detrimental to the host. Anthelmintic resistance (AR) against most anthelmintic drug classes is now widespread amongst the trichostrongyloids. Baseline establishment, followed by regular monitoring of the level of AR, is necessary for farmers and veterinarians to make informed decisions about parasite management. The detection of single nucleotide polymorphisms (SNPs) is a sensitive method to detect AR against benzimidazoles (BZs), one of the most widely used anthelmintic classes. Alpine transhumance constitutes a special type of pasturing of sheep from many different farms, the aim of this study was to investigate the prevalence of benzimidazole resistance alleles in this particular management system.ResultsSixteen sheep flocks in Styria and Salzburg in Austria were examined by pyrosequencing for SNPs at codons 167, 198 and 200 of the isotype-1 β-tubulin gene. The frequency of the resistance-associated exchange F200Y was 87–100% for H. contortus, 77–100% for T. colubriformis and < 5–66% for T. circumcincta. Additionally, the F167Y polymorphism was detected in T. colubriformis from two farms at a frequency of 19 and 23% respectively.ConclusionsThe high resistance allele frequency in H. contortus and T. colubriformis in the examined sheep population urgently calls for the development of new treatment strategies to sustainably control trichostrongyloid infections for this kind of pasturing, since the frequent mixing of flocks during the alpine summer grazing must be considered an important risk factor for the spread of resistant nematodes to a large number of farms.

Biocomputing label-free security system based on homogenous ligation chain reaction-induced dramatic change in melting temperature for screening single nucleotide polymorphisms

The development of smart platform with accurate, inexpensive and reliable detection of single-nucleotide polymorphisms (SNPs) has long been concerned in the fields of medical diagnosis and basic research. Here, we present a ligation chain reaction (LCR)-based sensing system for the cost-effective screening of SNPs by simply conducting DNA melting analysis. No chemical modification is required and the signaling operation is accomplished in homogeneous solution, circumventing the complex modification process and possibly compromised enzymatic activity associated with heterogeneous materials, such as quantum dot (QD) and gold nanoparticle (GNP). Due to the enzymatic catalysis and high fidelity of ligase, the system is capable of executing signal amplification, providing a high sensitivity and selectivity. KRAS gene is easily recognized and the site-specific mutation of guanine (G) to adenine (A), thymine (T) or cytosine (C) is accurately screened. Moreover, the excellent reliability was demonstrated by blind test and recovery test. LCR-based signaling mechanism was further used to develop the biocomputing security system, and two logic gates consisting of four single-stranded DNAs (ssDNAs) offer a double insurance to protect the information against illegal invasion, guaranteeing the reliability of output information. Once in the absence of one essential factor, the security system was always locked regardless of target key, serving as a novel strategy to ensure the safety of output information at molecular level. As a proof-of-concept scheme, this contribution introduces new insight into the development of DNA security systems and the exploitation of powerful signal transduction strategy suitable for rapid and convenient disease diagnosis.

Molecular inversion probe-rolling circle amplification with single-strand poly-T luminescent copper nanoclusters for fluorescent detection of single-nucleotide variant of SMN gene in diagnosis of spinal muscular atrophy

In this study, a simple fluorescent detection of survival motor neuron gene (SMN) in diagnosis of spinal muscular atrophy (SMA) based on nucleic acid amplification test and the poly-T luminescent copper nanoclusters (CuNCs) was established. SMA is a severely genetic diseases to cause infant death in clinical, and detection of SMN gene is a powerful tool for pre- and postnatal diagnosis of this disease. This study utilized the molecular inversion probe for recognition of nucleotide variant between SMN1 (c.840 C) and SMN2 (c.840 C > T) genes, and rolling circle amplification with a universal primer for production of poly-T single-strand DNA. Finally, the fluorescent CuNCs were formed on the poly-T single-strand DNA template with addition of CuSO4 and sodium ascorbate. The fluorescence of CuNCs was only detected in the samples with the presence of SMN1 gene controlling the disease of SMA. After optimization of experimental conditions, this highly efficient method was performed under 50 °C for DNA ligation temperature by using 2U Ampligase, 3 h for rolling circle amplification, and the formation of the CuNCs by mixing 500 μM Cu2+ and 4 mM sodium ascorbate. Additionally, this highly efficient method was successfully applied for 65 clinical DNA samples, including 4 SMA patients, 4 carriers and 57 wild individuals. This label-free detection strategy has the own potential to not only be a general method for detection of SMN1 gene in diagnosis of SMA disease, but also served as a tool for detection of other single nucleotide polymorphisms or nucleotide variants in genetic analysis through designing the different sensing probes.

Detection of Single Nucleotide Polymorphisms (SNPs) for Genes Cause Drug-Resistant in Iraqi Mycobacterium Tuberculosis isolates by new Pyrophosphate Technique.

In this search, a new pyrophosphate technique was proved. The technique was employed to single- nucleotide polymorphisms (SNPs), which diagnosis using a one-base extension reaction. Three Mycobacterium tuberculosis genes were chosen (Rpob, InhA, KatG) genes. Fifty-four specimens were used in this study fifty-three proved as drug-resistant specimens by The Iraqi Institute of Chest and Respiratory Diseases in Baghdad.; also one specimen was used as a negative control. The steps of this technique were by used a specific primer within each aliquot that has a short 3-OH end of the base of the target gene that was hybridized to the single-stranded DNA template. Then, the Taq polymerase enzyme and one of either α-thio-dATP, dTTP, dGTP, or dCTP were supplemented and incubated for 1 min. ATP is synthesis by convert Pyrophosphate freed by DNA polymerase using pyruvate phosphate dikinase (PPDK), and the amount of ATP estimates by the firefly luciferase reaction. This technique, which does not demand expensive equipment, can be applied to rapidly monitor a one-point mutation in the gene that causes drug-resistant in mycobacterium tuberculosis. The results showed a high variation in values of ATP formation through matching and mismatch bases added. So, this assay (which required only five minutes), enable to find the gene SNP causes resistant for the specific drug.

Development of two different pharmacogenomics detection methods for second-generation antipsychotic drugs quetiapine and aripiprazole

Abstract Objective: Second-generation antipsychotics are widely used in mental illness, but the treatment effects and side effects are affected by single nucleotide polymorphisms (SNPs) of related genes. Quetiapine and aripiprazole are two frequently used second-generation antipsychotic drugs. The aim of this study was to develop two different SNP detection methods for four SNP alleles associated with the pharmacokinetics of quetiapine and aripiprazole, based on high-resolution melting (HRM) and multicolor melting curve assay (MMCA) respectively. Methods: Whole genome DNA samples were obtained from 240 healthy people (107 females and 133 males) without genetic diseases. HRM methods were established using four kinds of specific primers and a saturated fluorescent dye. Each SNP allele with their own primers was detected in a single reaction. In the MMCA method, a multiplex polymerase chain reaction with 4 different-colored fluorescent probes was established to detect four SNP alleles in a single reaction. All experimental protocols were approved by the Ethics Committee of the Shanghai Children's Medical Center, China (SCMC-201015) on November 22, 2010. Results: Two detection methods for the pharmacogenomics of quetiapine and aripiprazole, based on HRM and MMCA respectively, were established in this study. The single-target HRM method can be completed in 96 minutes, whereas the quadruplex MMCA method takes 133 minutes. It was found that the results of HRM and MMCA for the four SNP alleles had 100% coincidence with Sanger sequencing in the 240 samples. Conclusion: This study developed two methods for the detection of four pharmacogenomic SNP alleles that correlated with quetiapine and aripiprazole. Both methods are rapid, cost-saving, highly accurate and potentially facilitate rational use of second-generation antipsychotics for clinical medication.

Ferrocene-labeled and purification-free electrochemical biosensor based on ligase chain reaction for ultrasensitive single nucleotide polymorphism detection

Single nucleotide polymorphisms (SNPs) are crucial during the early diagnosis of a given disease as well as in evaluating their response to certain drugs. Thus, this study sought the development of ferrocene (Fc)-labeled electrochemical biosensor for SNP detection. This proposed system involves the ligation of four short probes (e.g., A, B, A′, and B′, where B′ is labeled with an Fc-tag) in the presence of target DNA via ligase chain reaction (LCR), resulting in the formation of Fc-tagged duplex AB-A′B′ in 2n. Subsequently, immobilization of the Fc-tagged duplex AB-A′B′ on a single-stranded DNA capture probe (SC-DNA)-carboxyl multi-wall carbon nanotube (MWCNT-COOH) modified glassy carbon electrode (GCE) was accomplished through hybridization. Owing to the specificity of hybridization, and the use of Fc as electrochemical probe for detection of duplex AB-A′B′, such strategy realized directly analysis of LCR products without the need for purification. By taking advantage of the thermal stability and high-discrimination ability of HiFi Taq DNA ligase for single-base differences, the specificity of hybridization, the EGFR T790 M mutant DNA (MT-DNA) biosensor was developed to offer a low limit of detection (0.75 aM), a high discrimination of single-base mismatches [as low as 0.01% (molar fraction)], a wide linear range of more than 7 orders of magnitude (1 aM–10 pM), and the recovery rates (95.3%–107.8%) from human serum samples. Thus, the biosensor under development was found to be economical, highly-sensitive, and exceptionally selective for detection of SNPs, and as well as extending the versatile applications of LCR to offer great potential for diagnosis and individual clinical regimens.

A Fast and Scalable Workflow for SNPs Detection in Genome Sequences Using Hadoop Map-Reduce.

Next generation sequencing (NGS) technologies produce a huge amount of biological data, which poses various issues such as requirements of high processing time and large memory. This research focuses on the detection of single nucleotide polymorphism (SNP) in genome sequences. Currently, SNPs detection algorithms face several issues, e.g., computational overhead cost, accuracy, and memory requirements. In this research, we propose a fast and scalable workflow that integrates Bowtie aligner with Hadoop based Heap SNP caller to improve the SNPs detection in genome sequences. The proposed workflow is validated through benchmark datasets obtained from publicly available web-portals, e.g., NCBI and DDBJ DRA. Extensive experiments have been performed and the results obtained are compared with Bowtie and BWA aligner in the alignment phase, while compared with GATK, FaSD, SparkGA, Halvade, and Heap in SNP calling phase. Experimental results analysis shows that the proposed workflow outperforms existing frameworks e.g., GATK, FaSD, Heap integrated with BWA and Bowtie aligners, SparkGA, and Halvade. The proposed framework achieved 22.46% more efficient F-score and 99.80% consistent accuracy on average. More, comparatively 0.21% mean higher accuracy is achieved. Moreover, SNP mining has also been performed to identify specific regions in genome sequences. All the frameworks are implemented with the default configuration of memory management. The observations show that all workflows have approximately same memory requirement. In the future, it is intended to graphically show the mined SNPs for user-friendly interaction, analyze and optimize the memory requirements as well.

ReferenceSeeker: rapid determination of appropriate reference genomes

The large and growing number of microbial genomes available in public databases makes the optimal selection of reference genomes necessary for many in-silico analyses, e.g. single nucleotide polymorphism detection, scaffolding and comparative genomics, increasingly difficult. Here, we present ReferenceSeeker, a novel command line tool combining a fast kmer profile-based database lookup of candidate reference genomes with subsequent calculation of highly specific average nucleotide identity (ANI) values for the rapid determination of appropriate reference genomes. Pre-built databases for bacteria, archaea, fungi, protozoa and viruses based on the RefSeq database are provided for download. Availability and Implementation: ReferenceSeeker is open source software implemented in Python. Source code and binaries are freely available for download at https://github.com/oschwengers/referenceseeker​ under the GNU GPL3 license.

Detection of Single Nucleotide Polymorphisms in IL-1B gene in Iraqi Patients with Hepatitis B

Host genetic factors play an important role in the pathogenesis of hepatitis B virus (HBV) infection. However, the role of interleukin 1 receptor, type I (IL-1B) gene in HBV infection and breakthrough infection in people remains unclear. The aim of this study was to detect a SNP in IL-1B in HBV infection in Iraqi people.

The origin and current situation of Fusarium oxysporum f. sp. cubense tropical race 4 in Israel and the Middle East

Fusarium oxysporum f.sp. cubense (Foc) is considered one of the most devastating soilborne fungal pathogens of banana worldwide. Foc causing mortality to Cavendish group bananas, and belonging to the unique vegetative compatibility group (VCG) 01213/16 has been termed tropical race 4 (TR4) and has currently been renamed F. odoratissimum. The pathogen that was first detected approximately 50 years ago in South East Asia, has since spread to countries within the greater Mekong subregion and to Australia. Recently, the pathogen disseminated to India, Pakistan, Oman and Mozambique (Africa) and was identified in the South American continent in Colombia in 2019. In the Middle East, TR4 was first reported from Jordan and Lebanon, and later from Israel in 2016. In Israel, the pathogen was identified as TR4 by VCG tests, pathogenicity assays and molecular verification. The complete genomes of five representative TR4 isolates including two from Israel, one from Jordan, one from the Philippines, and one from Indonesia were sequenced, and single nucleotide polymorphisms (SNPs) analyses were conducted. SNPs were compared to 11 additional sequenced TR4 isolates, to determine the origin of the Israeli isolates. SNP detection and phylogeographical analyses determined that the Middle Eastern isolates are closely related, indicating that the pathogen most likely spread to Israel from Jordan, while those from Colombia are related to a representative isolate from Indonesia.

Using gold nanoparticles to detect single-nucleotide polymorphisms: toward liquid biopsy.

The possibility of detecting genetic mutations rapidly in physiological media through liquid biopsy has attracted the attention within the materials science community. The physical properties of nanoparticles combined with robust transduction methods ensure an improved sensitivity and specificity of a given assay and its implementation into point-of-care devices for common use. Covering the last twenty years, this review gives an overview of the state-of-the-art of the research on the use of gold nanoparticles in the development of colorimetric biosensors for the detection of single-nucleotide polymorphism as cancer biomarker. We discuss the main mechanisms of the assays that either are assisted by DNA-based molecular machines or by enzymatic reactions, summarize their performance and provide an outlook towards future developments.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection of the E198A SNP in the isotype-1 β-tubulin gene of Haemonchus contortus populations in China

Haemonchus contortus is one of the most important gastrointestinal nematodes of small ruminants around the world, seriously hampering the healthy development of the sheep industry. The control of this parasite mainly depends on anthelmintics, however, drug resistance of H. contortus has become a serious problems worldwide. Previous studies demonstrated that the E198A (GAA to GCA), a single nucleotide polymorphism (SNP) in the isotype-1 β-tubulin gene is associated with benzimidazole resistance in H. contortus. However, only PCR-RFLP and ARMS-PCR methods have been previously used for the detection of the E198A mutation. In the present study, a loop-mediated isothermal amplification (LAMP) assay was established for rapid detection of the E198A SNP in H. contortus. The results showed that optimization of LAMP reaction reagents and conditions could achieve this. The resulting amplicons were visualized by adding hydroxynaphthol blue dye (HNB) prior to amplification. The color of LAMP products amplified without DNA or from DNA from worms with the E198A homozygous susceptible genotype was still violet, but the products with DNA from worms with the E198A heterozygous genotype or the E198A resistant homozygous genotype changed to sky blue. The specificity of this method was further verified by sequencing, which confirmed the successful LAMP detection of the E198A mutation with high specificity. In conclusion, the developed LAMP method has high specificity and good reproducibility for screening the E198A SNP of isotype-1 β-tubulin gene of H. contortus of field samples without using sophisticated equipment, providing useful technique for the rapid detection and thus prevention and control of benzimidazole resistant H. contortus infections.

Solid-Phase Microextraction Enables Isolation of BRAF V600E Circulating Tumor DNA from Human Plasma for Detection with a Molecular Beacon Loop-Mediated Isothermal Amplification Assay.

Circulating tumor DNA (ctDNA) is a promising biomarker that can provide a wealth of information regarding the genetic makeup of cancer as well as provide a guide for monitoring treatment. Methods for rapid and accurate profiling of ctDNA are highly desirable in order to obtain the necessary information from this biomarker. However, isolation of ctDNA and its subsequent analysis remains a challenge due to the dependence on expensive and specialized equipment. In order to enable widespread implementation of ctDNA analysis, there is a need for low-cost and highly accurate methods that can be performed by nonexpert users. In this study, an assay is developed that exploits the high specificity of molecular beacon (MB) probes with the speed and simplicity of loop-mediated isothermal amplification (LAMP) for the detection of the BRAF V600E single-nucleotide polymorphism (SNP). Furthermore, solid-phase microextraction (SPME) is applied for the successful isolation of clinically relevant concentrations (73.26 fM) of ctDNA from human plasma. In addition, the individual effects of plasma salts and protein on the extraction of ctDNA with SPME are explored. The performed work expands the use of MB-LAMP for SNP detection as well as demonstrates SPME as a sample preparation tool for nucleic acid analysis in plasma.

High accuracy of HLA-B*27 genotyping by allele-specific real-time polymerase chain reaction in a heterogeneous population compared to flow cytometry and single nucleotide polymorphism detection assays

HLA-B27 is a risk marker for ankylosing spondylitis and other associated seronegative spondyloarthropathies. We compared three methods of HLA-B*27 typing in a New South Wales (NSW) population: flow cytometry, rs4349859 single nucleotide polymorphism (SNP) detection assay, and allele-specific real-time polymerase chain reaction (RT-PCR) analysis of exons 2 and 3. Over a 5-month period, 543 samples underwent flow cytometric testing and RT-PCR high-resolution melt analysis of rs4349859 SNP and of exon 2 (5' fragment) and exon 3. In the third method, positive samples were further analysed with fluorescent resonance emission transfer (FRET) RT-PCR of exon 2 fragments, 2a and 2b. HLA-B*27 and other genotypes were confirmed by Sanger sequencing of a 600 base pair fragment of exons 2 and 3. In our cohort, the rs4349859 SNP method had 78.6% sensitivity and 98.7% specificity. Screening with exon 2 (5' fragment) and exon 3 RT-PCR provided 100% sensitivity. Further testing with exon 2a and 2b FRET RT-PCR produced 100% specificity. This cascade approach with allele-specific RT-PCR assays was able to differentiate all samples into HLA-B*27 subtypes. HLA-B*27 genotyping with allele-specific RT-PCR assays, to screen for and confirm HLA-B27 positive samples, was more sensitive and specific than flow cytometry and rs4349859 SNP assays. It is a potentially cost-effective method for differentiating HLA-B27 subtypes. Our cascade genetic testing approach is suitable for replacing the current flow cytometric HLA-B27 assay for the heterogeneous NSW population.