Highly multiplex PCR assays by coupling the 5'-flap endonuclease activity of Taq DNA polymerase and molecular beacon reporters.

Real-time PCR is the most utilized nucleic acid testing tool in clinical settings. However, the number of targets detectable per reaction are restricted by current modes. Here, we describe a single-step, multiplex approach capable of detecting dozens of targets per reaction in a real-time PCR thermal cycler. The approach, termed MeltArray, utilizes the 5'-flap endonuclease activity of Taq DNA polymerase to cleave a mediator probe into a mediator primer that can bind to a molecular beacon reporter, which allows for the extension of multiple mediator primers to produce a series of fluorescent hybrids of different melting temperatures unique to each target. Using multiple molecular beacon reporters labeled with different fluorophores, the overall number of targets is equal to the number of the reporters multiplied by that of mediator primers per reporter. The use of MeltArray was explored in various scenarios, including in a 20-plex assay that detects human Y chromosome microdeletions, a 62-plex assay that determines Escherichia coli serovars, a 24-plex assay that simultaneously identifies and quantitates respiratory pathogens, and a minisequencing assay that identifies KRAS mutations, and all of these different assays were validated with clinical samples. MeltArray approach should find widespread use in clinical settings owing to its combined merits of multiplicity, versatility, simplicity, and accessibility.

Accurate Detection of Multiple Tumor Mutations in Formalin-Fixed Paraffin-Embedded Tissues by Coupling Sequence Artifacts Elimination and Mutation Enrichment With MeltArray.

Formalin-fixed paraffin-embedded (FFPE) tissues are the primary source of DNA for companion diagnostics (CDx) of cancers. Degradation of FFPE tissue DNA and inherent tumor heterogeneity constitute serious challenges in current CDx assays. To address these limitations, we introduced sequence artifact elimination and mutation enrichment to MeltArray, a highly multiplexed PCR approach, to establish an integrated protocol that provides accuracy, ease of use, and rapidness. Using PIK3CA mutations as a model, we established a MeltArray protocol that could eliminate sequence artifacts completely and enrich mutations from 23.5- to 59.4-fold via a single-reaction pretreatment step comprising uracil-DNA-glycosylase excision and PCR clamping. The entire protocol could identify 13 PIK3CA hotspot mutations of 0.05% to 0.5% mutant allele fractions within 5 hours. Evaluation of 106 breast cancer and 40 matched normal FFPE tissue samples showed that all 47 PIK3CA mutant samples were from the cancer tissue, and no false-positive results were detected in the normal samples. Further evaluation of 105 colorectal and 40 matched normal FFPE tissue samples revealed that 11 PIK3CA mutants were solely from the cancer sample. The detection results of our protocol were consistent with those of the droplet digital PCR assays that underwent sequence artifact elimination. Of the 60 colorectal samples with next-generation sequencing results, the MeltArray protocol detected 2 additional mutant samples with low mutant allele fractions. We conclude that the new protocol provides an improved alternative to current CDx assays for detecting tumor mutations in FFPE tissue DNA.

Multiplex Asymmetric PCR by Combining the Amplification Refractory Mutation System with the Homo-Tag-Assisted Nondimer System.

Asymmetric PCR is widely used to produce single-stranded amplicons (ss-amplicons) for various downstream applications. However, conventional asymmetric PCR schemes are susceptible to events that affect primer availability, which can be exacerbated by multiplex amplification. In this study, a new multiplex asymmetric PCR approach that combines the amplification refractory mutation system (ARMS) with the homo-Tag-assisted nondimer system (HANDS) is described. ARMS-HANDS (A-H) PCR utilizes equimolar-tailed forward and reverse primers and an excess Tag primer. The tailed primer pairs initiate exponential symmetric amplification, whereas the Tag primer drives linear asymmetric amplification along fully matched strands but not one-nucleotide mismatched strands, thereby generating excess ss-amplicons. The production of ss-amplicons is validated using agarose gel electrophoresis, sequencing, and melting curve analysis. Primer dimer alleviation is confirmed by both the reduced Loss function value and a 20-fold higher sensitivity in an 11-plex A-H PCR assay than in an 11-plex conventional asymmetric PCR assay. Moreover, A-H PCR demonstrates unbiased amplification by its allele quantitative ability in correct identification of all 31 trisomy 21 samples among 342 clinical samples. A-H PCR is a new generation of multiplex asymmetric amplification approach with various applications, especially when sensitive and quantitative detection is required.

Single-tube protocol for culture-independent spoligotyping of Mycobacterium tuberculosis based on MeltArray.

IMPORTANCE: Spacer oligonucleotide typing (spoligotyping), the first-line genotyping assay for Mycobacterium tuberculosis (MTB), plays a fundamental role in the investigation of its epidemiology and evolution. In this study, we established a single-tube spoligotyping assay using MeltArray, a highly multiplex polymerase chain reaction (PCR) approach that runs on a real-time PCR thermocycler. The MeltArray protocol included an internal positive control, gyrB, to indicate the abundance of MTB via the quantification cycle and 43 spacers to identify the spoligotype via melting curve analysis. The entire protocol was completed in a single step within 2.5 hours. The lowest detectable copy number for the tested strains was 20 copies/reaction and thus sufficient for analyzing both culture and sputum samples. We conclude that MeltArray-based spoligotyping could be used immediately in low- and middle-income countries with a high tuberculosis burden, given its easy access, improved throughput, and potential applicability to clinical samples.

Highly Sensitive Detection of PIK3CA Mutations by Looping-Out Probes-Based Melting Curve Analysis.

PIK3CA mutations have important therapeutic and prognostic implications in various cancer types. However, highly sensitive detection of PIK3CA hotspot mutations in heterogeneous tumor samples remains a challenge in clinical settings. To establish a rapid PCR assay for highly sensitive detection of multiple PIK3CA hotspot mutations. We described a novel melting curve analysis-based assay using looping-out probes that can enrich target mutations in the background of excess wild-type and concurrently reveal the presence of mutations. The analytical and clinical performance of the assay were evaluated. The developed assay could detect 10 PIK3CA hotspot mutations at a mutant allele fraction of 0.05-0.5% within 2 h in a single step. Analysis of 82 breast cancer tissue samples revealed 43 samples with PIK3CA mutations, 28 of which were confirmed by Sanger sequencing. Further testing of 175 colorectal cancer tissue samples showed that 24 samples contained PIK3CA mutations and 19 samples were confirmed by Sanger sequencing. Droplet digital PCR supported that all mutation-containing samples undetected by sequencing contained mutations with a low allele fraction. The rapidity, ease of use, high sensitivity and accuracy make the new assay a potential screening tool for PIK3CA mutations in clinical laboratories.

Quantification of Isoniazid-Heteroresistant Mycobacterium tuberculosis Using Droplet Digital PCR.

The quantitative detection of drug-resistance mutations in Mycobacterium tuberculosis (MTB) is critical for determining the drug resistance status of a sample. We developed a drop-off droplet digital PCR (ddPCR) assay targeting all major isoniazid (INH)-resistant mutations. The ddPCR assay consisted of three reactions: reaction A detects mutations at katG S315; reaction B detects inhA promoter mutations; and reaction C detects ahpC promoter mutations. All reactions could quantify 1%-50% of mutants in the presence of the wild-type, ranging from 100 to 50,000 copies/reaction. Clinical evaluation with 338 clinical isolates yielded clinical sensitivity of 94.5% (95% confidence interval [CI] = 89.1%-97.3%) and clinical specificity of 97.6% (95% CI = 94.6%-99.0%) compared with the traditional drug susceptibility testing (DST). Further clinical evaluation using 194 nucleic acid-positive MTB sputum samples revealed clinical sensitivity of 87.8% (95% CI = 75.8%-94.3%) and clinical specificity of 96.5% (95% CI = 92.2%-98.5%) in comparison with DST. All the mutant and heteroresistant samples detected by the ddPCR assay but susceptible by DST were confirmed by combined molecular assays, including Sanger sequencing, mutant-enriched Sanger sequencing and a commercial melting curve analysis-based assay. Finally, the ddPCR assay was used to monitor longitudinally the INH-resistance status and the bacterial load in nine patients undergoing treatment. Overall, the developed ddPCR assay could be an indispensable tool for quantification of INH-resistant mutations in MTB and bacterial loads in patients.

The relationship between psychological contract and occupational wellbeing of mother-infant care helpers in Zhejiang Province.

BACKGROUND: Mother-infant care (MIC) helpers have become an indispensable part in hospital services. In order to stabilize the MIC workforce, it is essential for administrators to have a solid understanding of what may influence occupational wellbeing. This article aims to explore how demographic characteristics and psychological contract affect occupational wellbeing among MIC helpers in Zhejiang Province, China. METHODS: This is a quantitative, cross-sectional study with MIC helpers in obstetrics from 20 hospitals in Zhejiang Province. A questionnaire including demographic data, a psychological contract scale and an occupational wellbeing scale was used in this study. Multiple linear regression was conducted to investigate the relationships between demographic characteristics, psychological contract and occupational wellbeing. RESULTS: This study surveyed 260 MIC helpers and found out the mean score of the psychological contract was 4.38 and the mean score of the occupational wellbeing was 4.01. Monthly income and psychological contract were significant predictors of occupational wellbeing (F = 142.167, p < 0.001), which explained 62.1% of the total amount of variance in occupational wellbeing. Psychological contract was the most important predictor of occupational wellbeing. CONCLUSIONS: Administrators should pay attention to the effect of psychological contract on occupational wellbeing of the MIC helpers in China. Focusing on the inner needs should be considered as a strategy for stabilizing the team.

Using Social Media to Disseminate Effective Pain Treatments for Newborns During Needle-Related Painful Procedures in China.

Social media has become a powerful approach to disseminating evidence to knowledge users. The BSweet2Babies video was developed in multiple languages showing the effectiveness of sweet solutions, skin-to-skin care, and breastfeeding during newborn painful procedures. This study aimed to disseminate the BSweet2Babies video in Chinese through social media platform of WeChat in China; evaluate the reach, acceptability, and recommendation of the video; and assess viewers' previous knowledge and experience of using the 3 strategies and intention to use these strategies in the future. Multiple dissemination strategies were used to maximize views for a 6-month dissemination period. The video received 19 812 views, 4306 "thumbs," and 671 participants completed surveys. Of the survey respondents, 393 were parents. Most respondents did not know these strategies and did not use or help parents use any of them. More healthcare professionals than parents intended to use or advocate for sweet solutions and breastfeeding. More healthcare professionals rated that the 3 strategies were easy to apply in real-life situations, but more parents evaluated that the length of the video was too long. Social media in China can be a promising approach to disseminating evidence on neonatal procedural pain treatments to healthcare professionals and the public.

Chinese newborn screening for the incidence of G6PD deficiency and variant of G6PD gene from 2013 to 2017.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common X-linked enzymopathies caused by G6PD gene variant. We aimed to provide the characteristics of G6PD deficiency and G6PD gene variant distribution in a large Chinese newborn screening population. We investigated the prevalence of G6PD in China from 2013 to 2017. Then, we examined G6PD activity and G6PD gene in representative Chinese birth cohort to explore the distribution of G6PD gene variant in 2016. We then performed multicolor melting curve analysis to classify G6PD gene variants in 10,357 neonates with activity-confirmed G6PD deficiency, and DNA Sanger sequencing for G6PD coding exons if hot site variants were not found. The screened population, organizations, and provinces of G6PD deficiency were increased from 2013 to 2017 in China. The top five frequency of G6PD gene variants were c.1376G>T, c.1388G>A, c.95A>G, c.1024C>T, and c.871G>A and varied in different provinces, with regional and ethnic features, and four pathogenic variant sites (c.152C>T, c.290A>T, c.697G>C, and c.1285A>G) were first reported. G6PD deficiency mainly occurs in South China, and the frequency of G6PD gene variant varies in different regions and ethnicities.

Barriers and Facilitators to Effective Procedural Pain Treatments for Pediatric Patients in the Chinese Context: A Qualitative Descriptive Study.

PURPOSE: To explore nurse and physician leaders' perceptions of barriers and facilitators to using evidence-based procedural pain treatments (i.e., sweet solutions, breastfeeding, and topical anesthetics) for hospitalized infants and children in the Chinese context. DESIGN AND METHODS: A descriptive qualitative study was conducted at three pediatric inpatient surgical units in one hospital in China. Purposive sampling was used to recruit nurse/physician leaders who were engaged in the clinical management of the 3 units. Data collection included a focus group and individual interviews. The Consolidated Framework for Implementation Research (CFIR) was used to guide the analysis of the data. RESULTS: Ten participants attended the focus group and 13 took part in individual interviews. The findings highlight 41 implementation determinants, including two neutral influencing factors, 22 barriers, and 17 facilitators. These influencing factors aligned with the four CFIR domains and 25 of the 29 CFIR constructs. Common barriers to using evidence-based pain treatments across different contexts were identified, such as health care professionals' limited knowledge and misconceptions on pediatric pain management, no specific policies, low priority, heavy workload, staff shortage, and limited time. Unique determinants in the Chinese context were also identified, including parents' concerns of these new interventions, parent wrath, hierarchical managerial system, and lower authority of nurses. CONCLUSIONS: Multiple barriers as well as facilitators to using evidence-based pain management strategies were identified. PRACTICE IMPLICATIONS: The findings inform further development of implementation strategies and could be used as baseline data for comparing the barriers and facilitators evaluated during and after implementation.

Rapid Identification of Clinically Relevant Mycobacterium Species by Multicolor Melting Curve Analysis.

The sustained increase in the incidence of nontuberculous mycobacterial (NTM) infection and the difficulty in distinguishing these infections from tuberculosis constitute an urgent need for NTM species-level identification. The MeltPro Myco assay is the first diagnostic system that identifies 19 clinically relevant mycobacteria in a single reaction based on multicolor melting curve analysis run on a real-time PCR platform. The assay was comprehensively evaluated regarding its analytical and clinical performances. The MeltPro Myco assay accurately identified 51 reference mycobacterial strains to the species/genus level and showed no cross-reactivity with 16 nonmycobacterial strains. The limit of detection was 300 bacilli/ml, and 1% of the minor species was detected in the case of mixed infections. Clinical studies using 1,163 isolates collected from five geographically distinct health care units showed that the MeltPro Myco assay correctly identified 1,159 (99.7%) samples. Further testing with 94 smear-positive sputum samples showed that all samples were correctly identified. Additionally, the entire assay can be performed within 3 h. The results of this study confirmed the efficacy of this assay in the reliable identification of mycobacteria, suggesting that it might potentially be used as a screening tool in regions endemic for tuberculosis.

Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovars.

BACKGROUND: While Salmonella serotyping is of paramount importance for the disease intervention of salmonellosis, a fast and easy-to-operate molecular serotyping solution remains elusive. We have developed a multiplex ligation reaction based on probe melting curve analysis (MLMA) for the identification of 30 common Salmonella serovars. METHODS: Serovar-specific primers and probes were designed based on a comparison of gene targets (wzx and wzy encoding for somatic antigen biosynthesis; fliC and fljB for flagellar antigens) from 5868 Salmonella genomes. The ssaR gene, a type III secretion system component, was included for the confirmation of Salmonella. RESULTS: All gene targets were detected and gave expected Tm values during assay evaluation. Cross reactions were not demonstrated between the 30 serovars (n = 211), or with an additional 120 serovars (n = 120) and other Enterobacteriaceae (n = 3). The limit of identification for all targets ranged from using 1.2 ng/µL to 1.56 ng/µL of DNA. The intra- and inter-assay standard deviations and the coefficients of variation were no more than 0.5 °C and less than 1% respectively, indicating high reproducibility. From consecutive outpatient stool samples (n = 3590) collected over a 10-month period at 11 sentinel hospitals in Shenzhen, China, we conducted a multicenter study using the traditional Salmonella identification workflow and the MLMA assay workflow in parallel. From Salmonella isolates (n = 496, 13.8%) derived by both workflows, total agreement (kappa = 1.0) between the MLMA assay and conventional serotyping was demonstrated. CONCLUSIONS: With an assay time of 2.5 h, this simple assay has shown promising potential to provide rapid and high-throughput identification of Salmonella serovars for clinical and public health laboratories to facilitate timely surveillance of salmonellosis.

Translation and linguistic validation of the implementation leadership scale in Chinese nursing context.

AIM: To translate the Implementation Leadership Scale (ILS) into Chinese and evaluate how Chinese nursing staff and leaders understood and responded to the Chinese ILS. BACKGROUND: Leadership is a critical factor for implementing evidence-based practice. The ILS is a valid and reliable instrument to understand leadership for evidence-based practice; however, this scale or the other similar instrument does not exist in Chinese. METHODS: We followed the translation and cross-cultural validation guideline developed by Sousa and Rojjanasrirat. Translation included two forward and blind backward translations, and their comparisons. Two rounds of cognitive interview were used to evaluate the linguistic validity. RESULTS: The translation process took 12 months. In the forward and backward translations, 24 translation issues were identified, of which semantic equivalence issues were most frequent. Ten nurses participated in each round of cognitive interviews and 33 linguistic issues were found. The final Chinese ILS had seven significant adaptations to the original instrument. CONCLUSION: This study provided a deep understanding of using the ILS in the local context and laid the foundation for future psychometric statistical testing. IMPLICATIONS FOR NURSING MANAGEMENT: Implementation leadership scale could support organisational leadership development programmes and strategies to facilitate and support EBP implementation and sustainability.

McSpoligotyping, a One-Step Melting Curve Analysis-Based Protocol for Spoligotyping of Mycobacterium tuberculosis.

The direct repeat (DR) region in the Mycobacterium tuberculosis (MTB) genome is composed of highly polymorphic direct variant repeats, which are the basis of spacer oligonucleotide typing (spoligotyping) to study the population structure and epidemiology of M. tuberculosis However, the membrane hybridization-based detection format requires various post-PCR manipulations and is prone to carryover contamination, restricting its wide use in high-TB-burden and resource-limited countries. We developed a one-step spoligotyping protocol, termed McSpoligotyping, based on real-time PCR. The typing results can be generated within 3 h by a single step of DNA addition. When evaluated with a collection of 1,968 isolates of MTB, McSpoligotyping agreed 97.71% (1,923/1,968) by sample and 99.93% (84,568/84,624) by spacer with traditional spoligotyping. Sequencing results showed that McSpoligotyping was even more accurate than spoligotyping (99.34% versus 98.37%). Further exploration of the false results of McSpoligotyping revealed the presence of single-nucleotide polymorphisms in the DR region. We concluded that McSpoligotyping could be used in epidemiology studies of tuberculosis by taking advantage of the shortened procedure, ease of use, and compatibility of results with standard spoligotyping.

Highly Sensitive Detection of Isoniazid Heteroresistance in Mycobacterium tuberculosis by DeepMelt Assay.

Detection of heteroresistance of Mycobacterium tuberculosis remains challenging using current genotypic drug susceptibility testing methods. Here, we described a melting curve analysis-based approach, termed DeepMelt, that can detect less-abundant mutants through selective clamping of the wild type in mixed populations. The singleplex DeepMelt assay detected 0.01% katG S315T in 105M. tuberculosis genomes/µl. The multiplex DeepMelt TB/INH detected 1% of mutant species in the four loci associated with isoniazid resistance in 104M. tuberculosis genomes/µl. The DeepMelt TB/INH assay was tested on a panel of DNA extracted from 602 precharacterized clinical isolates. Using the 1% proportion method as the gold standard, the sensitivity was found to be increased from 93.6% (176/188, 95% confidence interval [CI] = 89.2 to 96.3%) to 95.7% (180/188, 95% CI = 91.8 to 97.8%) compared to the MeltPro TB/INH assay. Further evaluation of 109 smear-positive sputum specimens increased the sensitivity from 83.3% (20/24, 95% CI = 64.2 to 93.3%) to 91.7% (22/24, 95% CI = 74.2 to 97.7%). In both cases, the specificity remained nearly unchanged. All heteroresistant samples newly identified by the DeepMelt TB/INH assay were confirmed by DNA sequencing and even partially by digital PCR. The DeepMelt assay may fill the gap between current genotypic and phenotypic drug susceptibility testing for detecting drug-resistant tuberculosis patients.

Correction to: Spectrum of PAH gene variants among a population of Han Chinese patients with phenylketonuria from northern China.

Following publication of the original article [1], the authors reported an error in Table 3 on page 4. Variant No. 18 should be " p.Ser339Phe c.1016C>T " (as given in Number 117 of Additional file 2).

A comparison of the MeltPro® HPV Test with the Cobas® HPV Test for detecting and genotyping 14 high-risk human papillomavirus types.

The clinical performance of the newly developed MeltPro® HPV Test, based on multicolor melting curve analysis, was evaluated and compared with the commercially available Cobas® HPV Test for detection of HPV and genotyping of HPV-16 and HPV-18. A total of 1647 cervical samples were analyzed with both tests. The agreement values were 96.2% for HPV detection, 99.6% for HPV-16 identification, and 99.7% for HPV-18 identification. All genotyping results from MeltPro® HPV Test showed that HPV-52, HPV-58, and HPV-16 were the most common types in this study. Intra-laboratory reproducibility studies showed 97.8% agreement while inter-laboratory reproducibility studies showed 96.9% agreement for the MeltPro® HPV Test. The MeltPro® HPV Test and Cobas® HPV Test are highly correlative and are useful for monitoring HPV infection.

Spectrum of PAH gene variants among a population of Han Chinese patients with phenylketonuria from northern China.

BACKGROUND: Phenylketonuria (PKU), which primarily results from a deficiency of phenylalanine hydroxylase (PAH), is one of the most common inherited inborn errors of metabolism that impairs postnatal cognitive development. The incidence of various PAH variations differs by race and ethnicity. The aim of the present study was to characterize the PAH gene variants of a Han population from Northern China. METHODS: In total, 655 PKU patients and their families were recruited for this study; each proband was diagnosed both clinically and biochemically with phenylketonuria. Subjects were sequentially screened for single-base variants and exon deletions or duplications within PAH via direct Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). RESULTS: A spectrum of 174 distinct PAH variants was identified: 152 previously documented variants and 22 novel variants. While single-base variants were distributed throughout the 13 exons, they were particularly concentrated in exons 7 (33.3%), 11 (14.2%), 6 (13.2%), 12 (11.0%), 3 (10.4%), and 5 (4.4%). The predominant variant was p.Arg243Gln (17.7%), followed by Ex6-96A > G (8.3%), p.Val399 = (6.4%), p.Arg53His (4.7%), p.Tyr356* (4.7%), p.Arg241Cys (4.6%), p.Arg413Pro (4.6%), p.Arg111* (4.4%), and c.442-1G > A (3.4%). Notably, two patients were also identified as carrying de novo variants. CONCLUSION: The composition of PAH gene variants in this Han population from Northern China was distinct from those of other ethnic groups. As such, the construction of a PAH gene variant database for Northern China is necessary to lay a foundation for genetic-based diagnoses, prenatal diagnoses, and population screening.

Nitratireductor aestuarii sp. nov., a marine alphaproteobacterium isolated from an estuary.

Two bacterial strains, 2-2-12-1T and 2-2-12-2, were isolated from the estuary of the Jiulong River, south-east China. Cells were Gram-stain-negative, non-motile, short rods without flagella. Growth occurred at 25-45 °C, at pH 5.0-9.0 and with 0.5-2 % (w/v) NaCl. The bacteria were unable to reduce nitrate. The predominant fatty acids were C19 : 0 cyclo ω9c and C18 : 1ω7c. Phylogenetic analysis based on 16S rRNA gene sequences showed that both strains belong to the genus Nitratireductor, family Phyllobacteriaceae, class Alphaproteobacteria. Their closest neighbours were 'Nitratireductor lucknowense' DSM 24322 (96.3 and 96.5 % similarity, respectively) and Nitratireductor pacificus MCCC 1A01024T (96.2 and 96.3 % similarity, respectively). The DNA G+C contents of the two strains were 56.7 mol%. DNA-DNA hybridization between strain 2-2-12-1T and the two most closely related type strains revealed 57.3 and 52.3 % relatedness, respectively. Evidence from genotypic, chemotaxonomic and phenotypic data indicated that strains 2-2-12-1T and 2-2-12-2 represent a novel species of the genus Nitratireductor, for which the name Nitratireductor aestuarii sp. nov. is proposed. The type strain is 2-2-12-1T (=LMG 29090T=CGMCC 1.15320T).

Spoligotyping of Mycobacterium tuberculosis Complex Isolates by Use of Ligation-Based Amplification and Melting Curve Analysis.

We report here a ligation-based spoligotyping that can identify unamplified spacers in membrane-based spoligotyping due to asymmetric insertion of IS6110 in the direct repeat locus. Our typing yielded 84.4% (411/487) concordance with traditional typing and 100% (487/487) accuracy when confirmed by DNA sequencing.