Universal Primer Set Research Articles

DNA Sequencing-Based Monitoring Of Serum Predicts Clinical Relapse Before CT Imaging in Diffuse Large B-Cell Lymphoma

BackgroundDiffuse large B-cell lymphoma (DLBCL) is the most common lymphoma with >40,000 cases annually in the US. While most patients are cured by frontline immunochemotherapy, up to 20% recur and salvage therapy is poor. Relapse detection relies on CT imaging, which is hampered by radiation exposure, high costs, high false positive rates, and infrequently detects relapse prior to symptoms. Novel methods to detect early relapse may improve outcomes. The LymphoSIGHT™ platform is a high-throughput DNA sequencing method that can detect minimal residual disease (MRD) of lymphoid malignancies in peripheral blood (PB) (Faham et al. Blood 2012) with a sensitivity of one lymphoma cell per million leukocytes. We showed it can also detect circulating tumor DNA in the PB of DLBCL pts (Armand Br J Haematol 2013). We assessed the sensitivity and specificity of LymphoSIGHT™ to detect circulating tumor DNA in serum after frontline therapy in DLBCL. MethodsAll pts were newly diagnosed DLBCL undergoing frontline EPOCH-based chemotherapy. Post-therapy surveillance included clinic visits, PB samples, and contrast CT scans at 3, 4, 6, 12 and 12 mos intervals for the first 5 years, respectively. Pre-treatment lymph node biopsies and PB samples collected at initial diagnosis and during surveillance visits were sent for VDJ amplification and MRD sequencing assessment. Using universal primer sets, we amplified immunoglobulin heavy and kappa chain (IGH and IGK) variable, diversity, and joining gene segments from genomic DNA in tumor biopsy and PB samples. Amplified products were sequenced, and tumor-specific clonotypes were identified for each pt based on their high frequency within the B-cell repertoire in the lymph node. The presence of the tumor-specific clonotype was then quantitated in PB samples. ResultsTwenty-seven pts, treated between July 1996 and July 2005, had adequate DNA from pre-treatment FFPE lymph node biopsies, and 25 (92.6%) had a high-frequency clonal rearrangement (MRD) detected. The median pt age was 49 years (20-75) and an IPI risk groups included 8 low, 3 low-intermediate, 11 high-intermediate and 3 high. The pts had a median of 12 surveillance CT scans (range 1-13) and 221 serum samples analyzed for MRD. Two pts progressed early after cycles 1 and 6. The first pt had progressive disease by CT scan after cycle 1 and remained MRD positive. The second pt remained MRD positive at the end of cycle 6, at which time a CT showed CR-unconfirmed, and progressed one month later. In the remaining 23 pts who achieved CR, seven relapsed at a median of 20.3 (range 5.6-151.6) months. The 16 pts in sustained CR had a median follow-up of 99.7 months (range 3.0-136.3) and none were MRD positive post-treatment (specificity 100%; 88.9-100%). There were 0 false positives in 200 post-treatment samples tested (specificity 100%, 99-100%). Of 7 relapsing pts, 6 had serum samples within 1 year of recurrence. Of these, 5 pts had a positive MRD (range 3.2 to 11.2 months) (sensitivity 83%; 44-98%) at a median of 9.2 months (range 3.2 to 11.2 months) prior to a positive CT scan (Figure). One relapsing pt only had a serum sample obtained 18 mos before CT recurrence and it was MRD negative. ConclusionsOur data suggest that a DNA sequencing-based MRD test of serum can predict clinical relapse with high sensitivity and specificity in DLBCL prior to CT progression. MRD assay detection may significantly reduce the cost, inconvenience and toxicity of surveillance. Importantly, early detection of recurrence may significantly improve pt outcomes. Additional pts are being analyzed and updated results will be available.This work was supported by the Intramural Research Program of NCI at NIH and Sequenta, Inc.▪ Disclosures:Kong:Sequenta: Employment, Equity Ownership. Zheng:Sequenta: Employment, Equity Ownership. Willis:Sequenta: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Faham:Sequenta: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

A Comparative Analysis Of Next-Generation Sequencing and Real-Time Quantitative PCR For Minimal Residual Disease Detection In Follicular Lymphomas

BackgroundDetection of minimal residual disease (MRD) by t(14;18) Real-Time Quantitative (RQ) PCR is an important tool during clinical follow-up in patients (pts) with follicular lymphoma (FL). However, only the major breakpoint region (MBR) and minor cluster region (mcr) breakpoints have been currently exploited as MRD targets. Therefore, only 50% to 65% of pts can be assessed by t(14;18) RQ-PCR. Alternative targets such as the immunoglobulin heavy chain variable region (IGH) can be used only with limitations due to somatic hypermutation. IGH-based next-generation sequencing (NGS) might provide an alternative approach and provide increased sensitivity, specificity, accuracy and reproducibility. We performed a comparison of the two approaches in 29 FL pts. Patients and MethodsOverall, 206 samples (85 bone marrow, 114 peripheral blood, 6 stem cell aliquots and one lymph node sample) were investigated from 29 FL pts in which RQ-PCR based MRD analysis had been performed in the context of prospective clinical trials. Overall, 33 dx and 173 follow-up (FU) samples were analyzed. 23/29 pts had a PCR detectable t(14;18) rearrangement, 5 pts had a clonal IGH rearrangement only and one patient had no marker by consensus PCR. NGS was performed at Sequenta in South San Francisco. Using universal primer sets, we amplified IGH variable, diversity, and joining gene segments from genomic DNA. Amplified products were sequenced to obtain a high degree of coverage and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency in the dx sample and then quantitated in FU samples.Overall comparability of MRD results by RQ-PCR and NGS was assessed using correlation analysis. A positive/negative discordance between two results was defined as major when the positive result was >1E-05 and minor when ≤1E-05; a quantitative discordance was defined as the presence of two positive results with a quantitative discrepancy >1 log. ResultsAll 29 pts were evaluable with at least one method. IGH NGS identified an index clone in 22/29 cases. Seven patients pts were only quantifiable by t(14;18) RQ-PCR while IGH NGS did not identify an index clone for sequencing. In all but one dx sample demonstrated low level lymphoma infiltration with MRD level below 10-3. In one of these cases, IG kappa could be successfully sequenced for MRD indicating that not only low level MRD but also somatic mutation of IGH is a potential pitfall for MRD detection by NGS in FL.15 pts were evaluable for MRD by t(14;18) RQ-PCR and NGS. In 97 FU samples, a significant concordance between MRD methods could be demonstrated (r2=0.80) (p<0.0001). Of these samples, 44 were MRD positive and 45 were MRD negative with both tools. A quantitative discordance occurred in 12/44 MRD positive samples, where MRD was higher in 7 samples and lower in 5 samples by NGS. A major discordance occurred in 4 samples where t(14;18) RQ-PCR was positive and NGS was negative. A minor discordance was detectable in 4 samples where in 2 samples t(14;18) RQ-PCR was positive and NGS was negative, and in the other 2 samples the opposite was correct.In 5 t(14;18) negative cases and one with an unusual large t(14;18) rearrangement MRD was quantified by IGH-RQ-PCR using cloned plasmids and IGH NGS. In all cases both methods also showed excellent concordance.One advantage of the IGH NGS approach is its ability to detect different IGH sequences generated by the somatic hypermutation process. We have seen in some patients a dynamic picture of the IGH clonotypes with the rise and fall of different clonotypes at different time points (Fig 1). [Display omitted] ConclusionsNGS represents a feasible tool for IGH-based MRD monitoring that allows analysis of a larger group of FL pts. Our results show that the two methods have a high level of correlation. Lymphoma infiltration of dx samples and somatic mutation of IGH is a critical point for identification of the tumor-specific clonotypes by NGS, therefore different MRD methods should complement each other to allow MRD assessment for the majority of pts. Furthermore IGH NGS sequencing has the potential to detect and track IGH evolution in FL. Disclosures:Kneba:Roche: Consultancy, Research Funding. Faham:Sequenta, Inc.: Employment, Patents & Royalties.

High-Throughput Sequencing Of T Cell Receptors Detects Signatures Of T Cell Repertoire That Predict MRD Status In Patients With Mantle Cell Lymphoma Undergoing Immunotransplantation

Background A clinical trial is ongoing at Stanford for MCL patients in first remission that interdigitates an autologous CpG-stimulated tumor cell vaccine with autologous peripheral blood stem cell transplant (PSCT) (NCT00490529). In this trial, blood samples collected before and after vaccination and serially post-transplant are assayed for minimal residual disease (MRD) and for T cell repertoire using the LymphoSIGHT™ sequencing method (Faham et al., Blood 2012). We identified a set of T cell clones that appear to be responding to the vaccine, and therefore we investigated whether the number of these clonotypes was correlated with MRD status. Methods Using universal primer sets, we amplified rearranged IgH variable (V), diversity, and joining (J) gene segments from genomic DNA. Amplified products were sequenced to obtain &gt;1 million reads. The B cell tumor-specific sequence was identified for each patient based on its high frequency in the original tumor biopsy. The presence of the tumor cells was then monitored in serial blood samples with a sensitivity of 1 cell per million leukocytes. The same blood samples were used for amplification, sequencing and analysis of the entire TCRβ repertoire. To facilitate identification of tumor vaccine-induced TCRβ clonotypes, we sequenced the TCRβ repertoire immediately before and after the administration of both the priming vaccination and a booster vaccination. We developed a metric called the vaccine response score (V score). This metric is calculated for each clonotype and reflects the increase in frequency after the initial vaccination AND after the boost. The formula for calculating V score is: V = F1 x F2 x square root [1/ (|F1 – F2| + 1)], where F1 and F2 represent the fold-change of the priming and boost vaccinations, respectively. Clonotypes with a V score &gt;10 were deemed to be vaccination-induced by virtue of these frequency changes. Results In a series of 12 vaccinated patients, the number of clonotypes with V score ≥ 10 ranged between 0 and 262, with a median of 57. We utilized an antigen-specific analysis to validate that clones with high V scores (≥ 10) were in fact tumor-specific. For this analysis, we incubated peripheral blood mononuclear cells (PBMCs) with the tumor and then sequenced the TCRβ repertoire from cells obtained after culture. Clones that were enriched after culture compared to pre-stimulation PBMCs were deemed to be antigen-specific. These clones that are antigen-specific are highly likely to have a high V score compared to a random frequency-matched set of clones (P two tailed = 1.8 x 10-10), providing further evidence that clones with a high V score are tumor-specific. We then analyzed the relationship between V score and clinical outcome. Patients could be stratified into two groups with “high” (&gt; 25) or “low” (&lt;25) numbers of vaccine-responsive clonotypes. Patients in the high V score group, who had larger numbers of putative tumor-specific T cells, were more likely to have sustained molecular remission during the first-year post-transplant compared with patients in the low V score group (P = 0.018) (Figure 1). Conclusions T cell repertoire analysis identified clonotypes responding to the vaccination, and the presence of these vaccine-specific clonotypes correlates with MRD positivity at the important landmark of one year post-PSCT. Further analysis of additional patients enrolled on the MCL trial is ongoing. This data underscores the prognostic relevance of the sequencing-based V score metric and provides a novel approach for assessment of cancer immunotherapy responses. Disclosures: Faham: Sequenta: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

A Cross-Sectional Study of Helicobacter Infection Among Laboratory Animals and Animal Research Workers

A Cross-Sectional Study of Helicobacter Infection Among Laboratory Animals and Animal Research Workers

Evaluating Detection Limits of Next-Generation Sequencing for the Surveillance and Monitoring of International Marine Pests

Most surveillance programmes for marine invasive species (MIS) require considerable taxonomic expertise, are laborious, and are unable to identify species at larval or juvenile stages. Therefore, marine pests may go undetected at the initial stages of incursions when population densities are low. In this study, we evaluated the ability of the benchtop GS Junior™ 454 pyrosequencing system to detect the presence of MIS in complex sample matrices. An initial in-silico evaluation of the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear small subunit ribosomal DNA (SSU) genes, found that multiple primer sets (targeting a ca. 400 base pair region) would be required to obtain species level identification within the COI gene. In contrast a single universal primer set was designed to target the V1–V3 region of SSU, allowing simultaneous PCR amplification of a wide taxonomic range of MIS. To evaluate the limits of detection of this method, artificial contrived communities (10 species from 5 taxonomic groups) were created using varying concentrations of known DNA samples and PCR products. Environmental samples (water and sediment) spiked with one or five 160 hr old Asterias amurensis larvae were also examined. Pyrosequencing was able to recover DNA/PCR products of individual species present at greater than 0.64% abundance from all tested contrived communities. Additionally, single A. amurensis larvae were detected from both water and sediment samples despite the co-occurrence of a large array of environmental eukaryotes, indicating an equivalent sensitivity to quantitative PCR. NGS technology has tremendous potential for the early detection of marine invasive species worldwide.

Molecular Identification of Field-CollectedCulicoidesLarvae in the Southern Part of Japan

Although Culicoides biting midges act as a vector of important human and domestic animal diseases, their ecology is poorly understood. The lack of proper identification systems of Culicoides larvae is one of the main obstacles to progress in research. Based on mitochondrial sequences of 19 Japanese Culicoides species, we designed a universal primer set to amplify the partial sequence of the mitochondrial cytochrome c oxidase I (cox 1). The polymerase chain reaction product amplified from extracted DNA of Culicoides larvae using the primer set was directly sequenced, and species identification based on the variation at cox1 was conducted. Using the molecular identification system, we sorted 243 specimens of field-collected larvae from the southern part of Japan into 10 species including Culicoides arakawae (Arakawa), Culicoides oxystoma Kieffer, and Culicoides brevitarsis Kieffer, which are regarded as vectors of important livestock animal diseases. Eight species of Culicoides larvae, including C. arakawae and C. oxystoma, were recovered from active paddy fields and an abandoned paddy field. The result suggests that paddy fields contribute to breeding a variety of Culicoides species and maintenance and spread of Culicoides-borne pathogens. In contrast, larvae of C. brevitarsis were collected from cattle dung in pastures. The molecular identification system described herein using nucleotide sequences successfully achieved larval identification and will be useful for a better understanding of larval habitats of Culicoides biting midges.

First Report of a 16SrIX Group ('Candidatus Phytoplasma phoenicium'-Related) Phytoplasma Associated with a Chrysanthemum Disease.

In November 2010, approximately 2% of the chrysanthemum (Chrysanthemum morifolium) cv. Paniz plants showed numerous small leaves in the top and stunting in a field collection of the National Research Center of Ornamental Plants in Mahallat, Iran. Next to these plants, some plants of the same collection showed leaves with a reddish and/or chlorotic discoloration around the veins. The observed symptoms were believed to represent infection by a phytoplasma and/or a viroid. Two plants with each type of the symptoms were individually analyzed. Using a total RNA extract from diseased leaves, RT-PCR with primer pairs targeting all known pospiviroids, including Chrysanthemum stunt viroid (CSVd) (3), were negative. Purified DNA was examined for the highly conserved phytoplasma 16S rRNA gene by nested-PCR using the universal primer sets P1/P7 and R16F2n/R16R2 (2). Fragments of 1.2 kb, obtained only from the plants with the small leaves and stunting, were sequenced and one of these sequences, which were identical, was deposited in GenBank (Accession No. KC176800). BLAST analysis of the chrysanthemum phytoplasma sequence exhibited 99% identity to Candidatus Phytoplasma phoenicium (Ca. P. phoenicium) species of the 16SrIX group. Subsequently, in silico RFLP analysis of the nested PCR product with the pDRAW32 program using AluI and TaqI restriction sites used for 16SrIX subgroups A, B, C, D, and E indicated that the 16SrIX chrysanthemum isolate belonged to subgroup D (1). Recently, based on GenBank sequences, several strains of Ca. P. phoenicium have been isolated and identified from diverse host species like Lactuca serriola, L. sativa, Solanum lycopersicon, Sonchus sp. [16SrIX-E], Carthamus tinctorius, and Prunus amygdalus [16SrIX-B] (4) in Iran. The vector species transmitting Ca. P. phoenicium to C. morifolium still needs to be identified. The leafhopper Neoaliturus fenestratus may be a potential vector as it is an often encountered efficient transmitter vector of 16SrIX group phytoplasmas in Iran (2). Next to the susceptibility of chrysanthemum to members of aster yellows, stolbur, and Ca. P. aurantifolia phytoplasma groups, this is, to our knowledge, the first report of a 16SrIX group member infecting chrysanthemum. The detection of this phytoplasma in chrysanthemum can form a new threat to this crop and other ornamentals in the Mahallat flower production region.

FIRST REPORT OF A “CANDIDATUS PHYTOPLASMA SOLANI” RELATED STRAIN ASSOCIATED WITH A DISEASE OF DATURA STRAMONIUM IN GREECE

Jimsonweed (Datura stramonium), family Solanaceae, a common weed in spring crops in Greece, includes two botanical varieties (var. stramonium and var. tatula). During a field trial at the Aristotle University Farm (40o32N’ 22o59’E, 6 m asl), in which four accessions of D. stramonium were tested for growth rate and alkaloid content, phytoplasma-like symptoms were observed. Initially, the affected plants showed interveinal chlorosis of the upper leaves, stunting and flower malformation, whereas at maturity they did not form normal fruits and developed leaf necrosis. To investigate the possibility of a phytoplasma infection, DNA was extracted from leaf samples of symptomatic and apparently healthy plants of both varieties according to Psifidi et al. (2010). A nested PCR was performed using two universal primer sets specific to the phytoplasma 16S rRNA gene: P1/P7 (Schneider et al., 1995) followed by R16F2n/R16R2 (Gundersen and Lee, 1996). The expected ca. 1,200 bp product, amplified esclusively from symptomatic plants of both varieties, was cloned and sequenced. BLAST analysis revealed 99% similarity with sequence AF248959 of stolbur phytoplasma (16SrXII group, Ca. Phytoplasma solani). Sequences from both varieties were identical, indicating infection by the same phytoplasma strain and were deposited in EMBL-EBI (accession Nos HE598778 and HE598779). Diseased plants of var. tatula and stramonium showed 49-69% and 38% reduction of the above-ground fresh weight, respectively, compared to healthy plants, hence making the phytoplasma an important pathogen of jimsonweed, which constitutes a reservoir plant in the field. This is, to our knowledge, the first report of a Ca. Phytoplasma solani-related disease in jimsonweed in Greece.

Quantitative detection of Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa in human oral epithelial cells from subjects with periodontitis and periodontal health

Epithelial cells in oral cavities can be considered reservoirs for a variety of bacterial species. A polymicrobial intracellular flora associated with periodontal disease has been demonstrated in buccal cells. Important aetiological agents of systemic and nosocomial infections have been detected in the microbiota of subgingival biofilm, especially in individuals with periodontal disease. However, non-oral pathogens internalized in oral epithelial cells and their relationship with periodontal status are poorly understood. The purpose of this study was to detect opportunistic species within buccal and gingival crevice epithelial cells collected from subjects with periodontitis or individuals with good periodontal health, and to associate their prevalence with periodontal clinical status. Quantitative detection of total bacteria and Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis in oral epithelial cells was determined by quantitative real-time PCR using universal and species-specific primer sets. Intracellular bacteria were visualized by confocal microscopy and fluorescence in situ hybridization. Overall, 33% of cell samples from patients with periodontitis contained at least one opportunistic species, compared with 15% of samples from healthy individuals. E. faecalis was the most prevalent species found in oral epithelial cells (detected in 20.6% of patients with periodontitis, P = 0.03 versus healthy individuals) and was detected only in cells from patients with periodontitis. Quantitative real-time PCR showed that high levels of P. aeruginosa and S. aureus were present in both the periodontitis and healthy groups. However, the proportion of these species was significantly higher in epithelial cells of subjects with periodontitis compared with healthy individuals (P = 0.016 for P. aeruginosa and P = 0.047 for S. aureus). Although E. faecalis and P. aeruginosa were detected in 57% and 50% of patients, respectively, with probing depth and clinical attachment level ≥6 mm, no correlation was found with age, sex, bleeding on probing or the presence of supragingival biofilm. The prevalence of these pathogens in epithelial cells is correlated with the state of periodontal disease.

Development and validation of a quantitative real-time polymerase chain assay for universal detection of the White Spot Syndrome Virus in marine crustaceans

BackgroundThe White Spot Syndrome Virus (WSSV), the sole member of the family Whispoviridae, is the etiological agent that causes severe mortality events in wild and farmed shrimp globally. Given its adverse effects, the WSSV has been included in the list of notifiable diseases of the Office of International Epizootic (OIE) since 1997. To date there are no known therapeutic treatments available against this lethal virus, and a surveillance program in brood-stock and larvae, based on appropriate diagnostic tests, has been strongly recommended. However, some currently used procedures intended for diagnosis of WSSV may be particularly susceptible to generate spurious results harmfully impacting the shrimp farming industry.MethodsIn this study, a sensitive one-step SYBR green-based real-time PCR (qPCR) for the detection and quantitation of WSSV was developed. The method was tested against several WSSV infected crustacean species and on samples that were previously diagnosed as being positive for WSSV from different geographical locations.ResultsA universal primer set for targeting the WSSV VP28 gene was designed. This method demonstrated its specificity and sensitivity for detection of WSSV, with detection limits of 12 copies per sample, comparable with the results obtained by other protocols. Furthermore, the primers designed in the present study were shown to exclusively amplify the targeted WSSV VP28 fragment, and successfully detected the virus in different samples regardless of their geographical origin. In addition, the presence of WSSV in several species of crustaceans, including both naturally and experimentally infected, were successfully detected by this method.ConclusionThe designed qPCR assay here is highly specific and displayed high sensitivity. Furthermore, this assay is universal as it allows the detection of WSSV from different geographic locations and in several crustacean species that may serve as potential vectors. Clearly, in many low-income import-dependent nations, where the growth of shrimp farming industries has been impressive, there is a demand for cost-effective diagnostic tools. This study may become an alternative molecular tool for a less expensive, rapid and efficient detection of WSSV.

Oral microbiome of deep and shallow dental pockets in chronic periodontitis.

We examined the subgingival bacterial biodiversity in untreated chronic periodontitis patients by sequencing 16S rRNA genes. The primary purpose of the study was to compare the oral microbiome in deep (diseased) and shallow (healthy) sites. A secondary purpose was to evaluate the influences of smoking, race and dental caries on this relationship. A total of 88 subjects from two clinics were recruited. Paired subgingival plaque samples were taken from each subject, one from a probing site depth >5 mm (deep site) and the other from a probing site depth ≤3mm (shallow site). A universal primer set was designed to amplify the V4–V6 region for oral microbial 16S rRNA sequences. Differences in genera and species attributable to deep and shallow sites were determined by statistical analysis using a two-part model and false discovery rate. Fifty-one of 170 genera and 200 of 746 species were found significantly different in abundances between shallow and deep sites. Besides previously identified periodontal disease-associated bacterial species, additional species were found markedly changed in diseased sites. Cluster analysis revealed that the microbiome difference between deep and shallow sites was influenced by patient-level effects such as clinic location, race and smoking. The differences between clinic locations may be influenced by racial distribution, in that all of the African Americans subjects were seen at the same clinic. Our results suggested that there were influences from the microbiome for caries and periodontal disease and these influences are independent.

A comparison between next-generation sequencing and ASO-qPCR for minimal residual disease detection in multiple myeloma.

8601 Background: Although molecular CR in multiple myeloma (MM) can be assessed by allele-specific oligonucleotide (ASO)-PCR, this technique requires preparation of clonotype-specific primers for each individual which is laborious and time-consuming. The usage of the LymphoSIGHT method, a next-generation sequencing (NGS)-based platform, may overcome these challenges and increase sensitivity and specificity. We compared the LymphoSIGHT approach with ASO-qPCR for minimal residual disease (MRD) detection in autografts in the autologous peripheral blood stem cell transplantation (ASCT) setting. Methods: Seventeen Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT were retrospectively analyzed. All patients had achieved a PR or CR after ASCT. Bone marrow (BM) slides from 13 MM patients and fresh BM cells from 4 MM patients at diagnosis as well as autografts were obtained for DNA extraction. Using universal primer sets, we amplified IGH variable (V), diversity (D), and joining (J) gene segments, IGH-DJ, and IGK from genomic DNA. Amplified products were subjected to deep sequencing using NGS. Reads were analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency in BM samples. The presence of the myeloma clonotype was then assessed in follow-up samples. Results: MRD in autografts was detected in 6 of 17 (35%) by ASO-qPCR and 13 of 17 (76%) by NGS. When MRD was assessed by NGS, 6 MRD (+) cases received post-ASCT therapy while 4 MRD (-) cases and 7 MRD (+) cases were followed without post-ASCT therapy. The MRD (-) cases tended to show a better PFS than the MRD (+) cases with post-ASCT therapy (P = 0.26) and those without post-ASCT therapy (P = 0.09) although overall survival rates were comparable among the three groups. There was no difference in PFS between MRD (-) and MRD (+) cases when MRD was assessed by ASO-qPCR (P = 0.6). These studies will be extended in 30 additional MM patients, and results will be presented. Conclusions: MRD-negativity in autografts revealed by NGS may be more closely associated with durable remission of MM than that revealed by ASO-qPCR.

Rapid, Simple and Sensitive Detection of Q Fever by Loop-Mediated Isothermal Amplification of the htpAB Gene

BackgroundQ fever is the most widespread zoonosis, and domestic animals are the most common sources of transmission. It is not only difficult to distinguish from other febrile diseases because of the lack of specific clinical manifestations in humans, but it is also difficult to identify the disease in C. burnetii-carrying animals because of the lack of identifiable features. Conventional serodiagnosis requires sera from the acute and convalescent stages of infection, which are unavailable at early diagnosis. Nested PCR and real-time PCR require equipment. In this study, we developed a Loop-Mediated Isothermal Amplification (LAMP) assay to identify C. burnetii rapidly and sensitively.MethodsA universal LAMP primer set was designed to detect the repeated sequence IS1111a of the htpAB gene of C. burnetii using PrimerExplorer V4 software. The sensitivity of the LAMP assay was evaluated using known quantities of recombined reference plasmids containing the targeted genes. The specificity of the developed LAMP assay was determined using 26 members of order Rickettsiae and 18 other common pathogens. The utility of the LAMP assay was further compared with real time PCR by the examination 24 blood samples including 6 confirmed and 18 probable Q fever cases, which diagnosed by IFA serological assessment and real time PCR. In addition, 126 animal samples from 4 provinces including 97 goats, 7 cattle, 18 horses, 3 marmots and 1 deer were compared by these two methods.ResultsThe limits of detection of the LAMP assay for the htpAB gene were 1 copy per reaction. The specificity of the LAMP assay was 100%, and no cross-reaction was observed among the bacteria used in the study. The positive rate of unknown febrile patients was 33.3%(95%CI 30.2%–36.4%) for the LAMP assay and 8.3%(95%CI 7.4%–9.2%) for the real time PCR(P<0.05). Similarly, the total positive rate of animals was 7.9%(95%CI 7.1%–8.7%) for the LAMP assay and 0.8%(95%CI 0.7%–0.9%)for the real time PCR(P<0.01). Using the developed LAMP assay, Q fever in the Yi Li area, Xinjiang Province, was confirmed.ConclusionsThe LAMP assay is a potential tool to support the diagnosis of Q fever in humans and domestic animals in the field, especially in the rural areas of China, because of its rapid and sensitive detection without the aid of sophisticated equipment or a complicated protocol.

Efficient Sequencing of Anuran mtDNAs and a Mitogenomic Exploration of the Phylogeny and Evolution of Frogs

Anura (frogs and toads) constitute over 88% of living amphibian diversity but many important questions about their phylogeny and evolution remain unresolved. For this study, we developed an efficient method for sequencing anuran mitochondrial DNAs (mtDNAs) by amplifying the mitochondrial genome in 12 overlapping fragments using frog-specific universal primer sets. Based on this method, we generated 47 nearly complete, new anuran mitochondrial genomes and discovered nine novel gene arrangements. By combining the new data and published anuran mitochondrial genomes, we assembled a large mitogenomic data set (11,007 nt) including 90 frog species, representing 39 of 53 recognized anuran families, to investigate their phylogenetic relationships and evolutionary history. The resulting tree strongly supported a paraphyletic arrangement of archaeobatrachian (=nonneobatrachian) frogs, with Leiopelmatoidea branching first, followed by Discoglossoidea, Pipoidea, and Pelobatoidea. Within Neobatrachia, the South African Heleophrynidae is the sister-taxon to all other neobatrachian frogs and the Seychelles-endemic Sooglossidae is recovered as the sister-taxon to Ranoidea. These phylogenetic relationships agree with many nuclear gene studies. The chronogram derived from two Bayesian relaxed clock methods (MultiDivTime and BEAST) suggests that modern frogs (Anura) originated in the early Triassic about 244 Ma and the appearance of Neobatrachia took place in the late Jurassic about 163 Ma. The initial diversifications of two species-rich superfamilies Hyloidea and Ranoidea commenced 110 and 133 Ma, respectively. These times are older than some other estimates by approximately 30-40 My. Compared with nuclear data, mtDNA produces compatible time estimates for deep nodes (>150 Ma), but apparently older estimates for more shallow nodes. Our study shows that, although it evolves relatively rapidly and behaves much as a single locus, mtDNA performs well for both phylogenetic and divergence time inferences and will provide important reference hypotheses for the phylogeny and evolution of frogs.

Sequencing Angiosperm Plastid Genomes Made Easy: A Complete Set of Universal Primers and a Case Study on the Phylogeny of Saxifragales

Plastid genomes are an invaluable resource for plant biological studies. However, the number of completely sequenced plant plastid genomes is still small compared with the vast number of species. To provide an alternative generalized approach, we designed a set of 138 pairs of universal primers for amplifying (termed “short-range PCR”) and sequencing the entire genomes of the angiosperm plastid genomes. The universality of the primers was tested by using species from the basal to asterid angiosperms. The polymerase chain reaction (PCR) success rate was higher than 96%. We sequenced the complete chloroplast genome of Liquidambar formosana as an example using this method and compared it to the genomes independently determined by long-range PCR (from 6.3 kb to 13.3 kb) and next-generation sequencing methods. The three genomes showed that they were completely identical. To test the phylogenetic efficiency of this method, we amplified and sequenced 18 chloroplast regions of 19 Saxifragales and Saxifragales-related taxa, as a case study, to reconstruct the phylogeny of all families of the order. Phylograms based on a combination of our data, together with those from GenBank, clearly indicate three family groups and three single families within the order. This set of universal primers is expected to accelerate the accumulation of angiosperm plastid genomes and to make faster mass data collection of plastid genomes for molecular systematics.

Development of Loop-Mediated Isothermal Amplification (LAMP) for Universal Detection of Enteroviruses

Enteroviruses are found in most environments and cause several diseases in humans. Loop-mediated isothermal amplification (LAMP) was adapted and evaluated for the rapid detection of enteroviruses. Based on the highly conserved 5' untranslated region (5'-UTR) of the human enteroviruses (HEVs), particularly human enterovirus A (HEV-A) and HEV-B, a set of universal primers was designed. The LAMP amplification was carried out under isothermal conditions at 61°C, depending on the template concentration results were obtained within 45-90min. The detection limits were found to be 10(1) copies of cloned enterovirus 71 fragments, more sensitive than conventional PCR. Nine water samples collected from drinking water sources during three seasons and 19 stool specimens collected from HFMD patients were analyzed. By using the LAMP assay, the majority of samples was tested positive, 9/9 (100%) and 18/19 (94.7%), respectively. LAMP is a practical method for the rapid detection of enteroviruses in environmental and clinical samples.

Universal DNA primers for amplification of complete mitochondrial protein-coding genes and ribosomal RNA genes from Crocodilia

PCR amplification and direct sequencing of mitochondrial genes of target taxa with universal primers designed according to a public DNA database can provide valuable information for phylogenetic inferences. This study describes the design of universal primer sets for an amplified partial 12S rRNA gene and fourteen other complete genes (16S rRNA gene plus 13 protein-coding genes) from the mitochondrial genome of 21 crocodilian species. This provides useful tools for the study of genetic variability in Crocodilia at or below the species level.

Hepatitis E virus isolated from rabbits is genetically heterogeneous but with very similar antigenicity to human HEV

Rabbit hepatitis E virus (HEV) in China may represent a novel HEV genotype, although no consensus has been reached. It is unclear whether the ORF2 capsid protein containing the immunodominant epitopes from rabbit HEV differs from those of human HEV. In this study, 661 bile samples collected from domestic rabbits in Jiangsu province, eastern China were amplified by RT-nPCR using a set of HEV universal ORF2 primers. All 42 (6.4%) positive PCR products were sequenced. Phylogenetic analysis using the ORF2 sequences of 557 bp in length showed the Jiangsu isolates were separate from HEV genotypes 1, 2, 3, 4, avian HEV and rat HEV, and clustered together with rabbit HEV sequences. These 42 isolates were divided into five branches including two newly identified in the present study. Comparison with rabbit HEV sequences from China available in GenBank, using a 298 bp ORF2 segment, showed these sequences clustered together into a unique rabbit HEV clade, and were divided into eight sub-branches with high genetic heterogeneity. In addition, 267 serum samples collected from domestic rabbits, serial serum samples from two rhesus monkeys experimentally infected with HEV genotype 1 or 4, and serial serum samples from two New-Zealand rabbits infected experimentally with rabbit HEV were tested simultaneously by EIA using recombinant truncated ORF2 capsid proteins derived from rabbit and human HEV. The virtually identical results obtained suggest that rabbit and human HEV ORF2 antigens contain very similar immunodominant epitopes. All these data are helpful to identify the biological characteristics of the newly identified rabbit HEV.

Application of peptide nucleic acid (PNA)-PCR clamping technique to investigate the community structures of rhizobacteria associated with plant roots

The contamination of plant organelle (mitochondria and plastid) genes in the DNA extraction step becomes a major drawback in investigating the community structures of bacteria associated with plant samples. This is because organelle small subunit ribosomal RNA (SSU rRNA) genes are easily amplified by polymerase chain reaction (PCR) with a set of universal primers for bacteria. To suppress the PCR amplification of the organelle SSU rRNA genes, a peptide nucleic acid (PNA)-PCR clamping technique was applied for selective amplification of bacterial SSU rRNA genes. The PNA oligomers, which had sequences that were complementary to mitochondria and plastid SSU rRNA genes, were designed to overlap the region in the 1492r primer-binding site. PCR with the PNA oligomers significantly suppressed the amplification of the organelle SSU rRNA genes from spinach and cucumber roots. Terminal restriction fragment length polymorphism (T-RFLP) analysis showed that the conventional amplification without PNA oligomers generated the predominant T-RFLP fragments derived from mitochondria and plastids, whereas there was little detection of the rhizobacterial fragments. In contrast, several other T-RFLP fragments derived from rhizobacteria were detected in the products amplified with PNA oligomers, thereby enabling us to differentiate the community structures in spinach and cucumber roots. Thus, application of PNA-PCR clamping was considered to be effective and is a useful technique to amplify the rhizobacterial SSU rRNA genes from selectively extracted DNA containing plant mitochondria and plastid genes.

First Report of Potato spindle tuber viroid Naturally Infecting Greenhouse Tomatoes in North Carolina.

In spring 2012, a severe disease was observed on a limited number of tomato plants (Solanum lycopersicum L.) in a research greenhouse facility in western North Carolina. The first symptoms noted were downward curling of the terminal leaves accompanied by a rough puckered darker green texture. This was followed in time by greater distortion of the leaves with pale green on leaf margins. Older leaves with symptoms developed necrosis, with necrotic spots and streaks appearing on a few fruits. On some of these affected fruits, stems, peduncles, pedicels, and sepals also showed symptoms. Infected plants were badly stunted, and fruits in the upper parts of plants displaying severe symptoms remained very small. In just a few months, the disease spread to other tomato plants inside the greenhouse. A survey in May 2012 showed a disease incidence of 18% (156 symptomatic plants out of a total of 864) in this greenhouse. Initial screenings for possible viruses using ELISA (Agdia, Elkhart, IN), as well as a reverse transcription (RT)-PCR panel of 15 common tomato viruses in our laboratory were negative. Because of the symptoms and negative results for viruses, a viroid infection was suspected. Total plant RNA was prepared using TRIzol reagent (Invitrogen, Carlsbad, CA) from leaf tissues of eight diseased plants and one seed sample. Using real-time RT-PCR developed against Potato spindle tuber viroid (PSTVd) and some related pospiviroids (1), positive signals were observed with a mean Ct = 13.24 for leaf tissues and Ct = 19.91 for the seed sample. To obtain a full viroid genome, RT-PCR using two different sets of primers, one specific for PSTVd (PSTVd-F and PSTVd-R) (2), and a universal primer set for pospiviroids (MTTVd-F and MTTVd-R) (3) was performed. RT-PCR generated amplicons with expected size of ~360 bp from all eight leaf and one seed samples, but not from a healthy control. PCR products were cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). A total of 22 full genomic sequences were obtained. A multi-sequence alignment generated a consensus sequence of 360 nt, designated as NC12-01 (GenBank Accession No. JX280944). BLASTn search in the NCBI database revealed the highest sequence identity of 96.9% to Australian (AY962324) and UK (AJ583449) isolates of PSTVd and 95.9% identity to the tomato isolate of PSTVd-CA1 (HM753555). Similar disease symptoms were observed on two 'Rutgers' tomato plants 2 weeks post mechanical inoculation and the presence of PSTVd was confirmed by real-time RT-PCR (1). A mock-inoculated plant did not show any symptoms. In the U.S., natural infection of PSTVd on tomato was first identified in California in 2010 (3). To our knowledge, this is the first report of a natural occurrence of PSTVd on tomato in the eastern U.S. The diseased plants were contained, properly disposed of, and eradicated in this location. The broader geographic distribution of PSTVd on tomato in the U.S., and the potential latent infection in potato and a number of ornamentals (4), emphasizes the need for better plant and seed health tests for viroids on these plants.