A New Reference Plasmid “pGMT27” Provides an Efficient Transgenic Detection Method for Flue-Cured Tobacco

Owing to the economic value of its foliage, tobacco (Nicotiana tabacum) is cultivated all across the world. For the detection of genetically modified (GM) tobacco, there is a lack of universal standard material which ultimately limits the detection methods because the accuracy and comparability of the results cannot be ensured. Here, we prepared a reference plasmid “pGMT27” for the detection of GM tobacco, which was 18,296 bp in length harboring two of the tobacco endogenous and seven exogenous genes. By using qualitative PCR test for the nine genes, 10 copies were used for plasmid sensitivity. In the quantitative real-time PCR (qPCR) assays with pGMT27 as a calibrator, the reaction efficiencies for P-35S and NR were 101.427% and 98.036%, respectively, whereas the limit of detection (LOD) and limit of quantification (LOQ) were 5 copies and 10 copies per reaction. For standard deviation (SD) and relative standard deviation (RSD) of the Ct values, the repeatability values were from 0.04 to 0.42 and from 0.18% to 1.29%, respectively; and the reproducibility values were from 0.04 to 0.39 and from 0.18% to 1.14%, respectively. For the unknown sample test, the average conversion factor (Cf) was 0.39, and the accuracy bias was from −15.55% to 1.93%; for precision, the SD values ranged from 0.02 to 0.62, while RSD values were from 1.34% to 10.6%. We concluded that using the pGMT27 plasmid as a calibrator provided a highly efficient transgenic detection method for flue-cured tobacco.

Accurate Quantification of AAV Vector Genomes by Quantitative PCR

The ability to accurately determine the dose of an adeno-associated viral (AAV) therapeutic vector is critical to the gene therapy process. Quantitative PCR (qPCR) is one of the common methods to quantify the AAV vector titre, but different variables can lead to inconsistent results. The aim of this study was to analyze the influence of the conformation of the DNA used as the standard control, and the enzymatic digestion was performed to release the viral genome from the protein capsid on the physical genome titration of a clinically relevant AAV8.RPGR vector, made to good laboratory practice standards in an academic setting. The results of this study showed that the conformation of the DNA used as standard has a significant impact on the accuracy of absolute quantification by qPCR. The use of supercoiled undigested plasmid DNA template generated a higher apparent titer, as compared to the use of linearized plasmid as the standard. In contrast to previous studies, the pre-treatment of the samples with Proteinase K, in addition to the high temperature step used after DNase I digestion, resulted in a reduction on AAV titers. Ideally, all AAV documentation should state which form of reference plasmid and which pre-treatment of the samples have been used to calculate titers, so that appropriate comparisons relating to dose toxicity and transduction efficacy can be made in the clinical scenario.

Reference Plasmid pHXB2_D is an HIV-1 Molecular Clone that Exhibits Identical LTRs and a Single Integration Site Indicative of an HIV Provirus

Objective: To compare long-read nanopore DNA sequencing (DNA-seq) with short-read sequencing-by-synthesis for sequencing a full-length (e.g., non-deletion, nor reporter) HIV-1 model provirus in plasmid pHXB2_D. Design: We sequenced pHXB2_D and a control plasmid pNL4-3_gag-pol(Δ1443-4553)_EGFP with long- and short-read DNA-seq, evaluating sample variability with resequencing (sequencing and mapping to reference HXB2) and de novo viral genome assembly. Methods: We prepared pHXB2_D and pNL4-3_gag-pol(Δ1443-4553)_EGFP for long-read nanopore DNA-seq, varying DNA polymerases Taq (Sigma-Aldrich) and Long Amplicon (LA) Taq (Takara). Nanopore basecallers were compared. After aligning reads to the reference HXB2 to evaluate sample coverage, we looked for variants. We next assembled reads into contigs, followed by finishing and polishing. We hired an external core to sequence-verify pHXB2_D and pNL4-3_gag-pol(Δ1443-4553)_EGFP with single-end 150 base-long Illumina reads, after masking sample identity. Results: We achieved full-coverage (100%) of HXB2 HIV-1 from 5' to 3' long terminal repeats (LTRs), with median per-base coverage of over 9000x in one experiment on a single MinION flow cell. The longest HIV-spanning read to-date was generated, at a length of 11,487 bases, which included full-length HIV-1 and plasmid backbone with flanking host sequences supporting a single HXB2 integration event. We discovered 20 single nucleotide variants in pHXB2_D compared to reference, verified by short-read DNA sequencing. There were no variants detected in the HIV-1 segments of pNL4-3_gag-pol(Δ1443-4553)_EGFP. Conclusions: Nanopore sequencing performed as-expected, phasing LTRs, and even covering full-length HIV. The discovery of variants in a reference plasmid demonstrates the need for sequence verification moving forward, in line with calls from funding agencies for reagent verification. These results illustrate the utility of long-read DNA-seq to advance the study of HIV at single integration site resolution.

Genetic Characterization of Multidrug-Resistant Salmonella Typhimurium Carrying Mcr-1 in China

Background: With the rapid emergence of plasmid mediated polymyxin resistance gene mcr-1, multidrug-resistant Salmonella caused great troubles in clinical treatment and attracted extensive attention. Here we report multidrug-resistant Salmonella strains harboring mcr-1 in China, which are resistant to both polymyxin, traditional antibiotics and even clinically wide-used antibiotics. Methods: We screened 1454 strains of Salmonella collected in our laboratory from 2006 to 2018 from 3 provinces or regions for mcr-1 by PCR. Antimicrobial susceptibility testing was determined. Plasmid conjugation assays were carried out to analysis the transferability of polymyxin resistance. Genetic polymorphism analysis of Salmonella was performed using the PFGE, and the plasmid profiles were characterized by S1-PFGE and southern blotting. The plasmids harboring mcr-1 were sequenced and compared. Results: Eleven S. Typhimurium isolates harboring mcr-1 with polymyxin resistance (MICs 4μg/ml) were identified from intestinal infections and foods in China. All S. Typhimurium isolates were multidrug-resistant to traditional antibiotics and even clinically wide-used antibiotics. Three types of plasmids harboring mcr-1 were recovered (IncHI2, IncX4 and IncI2). Compared with the reference plasmid, IncX4 and IncI2 plasmids had extremely similar typical backbone, and contain only mcr-1 resistance gene. However, IncHI2 were the most diverse type of plasmid due to containing a large MDR region, including blaCTX-M, oqxB, sul, aph, aadA and blaTEM. IncHI2 plasmids were observed to contain only one or no insertion sequence ISApl1 around mcr-1, without forming a circular intermediate. Conclusion: With the horizontal transfer of different types of plasmids, mcr-1 is widely spread worldwide. These prevalent plasmids are responsible for resistance to polymyxin, traditional antibiotics and even clinically wide-used antibiotics resulting from transmission of mcr-1 and other resistance genes. Our studying emphasizes the necessity to jointly monitor its international epidemic and preemptive further upgrade.

Full-coverage sequencing of HIV-1 provirus from a reference plasmid

ABSTRACTObjective(s)To evaluate nanopore DNA sequencing for sequencing full-length HIV-1 provirus.DesignI used nanopore sequencing to sequence full-length HIV-1 from a plasmid (pHXB2).MethodspHXB2 plasmid was processed with the Rapid PCR-Barcoding library kit and sequenced on the MinION sequencer (Oxford Nanopore Technologies, Oxford., UK). Raw fast5 reads were converted into fastq (base called) with Albacore, Guppy, and FlipFlop base callers. Reads were first aligned to the reference with BWA-MEM to evaluate sample coverage manually. Reads were then assembled with Canu into contigs, and contigs manually finished in SnapGene.ResultsI sequenced full-length HXB2 HIV-1 from 5’ to 3’ LTR (100%), with median per-base coverage of over 9000x in one 12-barcoded experiment on a single MinION flow cell. The longest HIV-spanning read to-date was generated, at a length of 11,487 bases, which included full-length HIV-1 and plasmid backbone on either side. At least 20 variants were discovered in pHXB2 compared to reference.ConclusionsThe MinION sequencer performed as-expected, covering full-length HIV. The discovery of variants in a dogmatic reference plasmid demonstrates the need for single-molecule sequence verification moving forward. These results illustrate the utility of long read sequencing to advance the study of HIV at single integration site resolution.

Development of a Novel Reference Plasmid for Accurate Quantification of Genetically Modified Kefeng6 Rice DNA in Food and Feed Samples

Reference plasmids are an essential tool for the quantification of genetically modified (GM) events. Quantitative real-time PCR (qPCR) is the most commonly used method to characterize and quantify reference plasmids. However, the precision of this method is often limited by calibration curves, and qPCR data can be affected by matrix differences between the standards and samples. Here, we describe a digital PCR (dPCR) approach that can be used to accurately measure the novel reference plasmid pKefeng6 and quantify the unauthorized variety of GM rice Kefeng6, eliminating the issues associated with matrix effects in calibration curves. The pKefeng6 plasmid was used as a calibrant for the quantification of Kefeng6 rice by determining the copy numbers of event- (77 bp) and taxon-specific (68 bp) fragments, their ratios, and their concentrations. The plasmid was diluted to five different concentrations. The third sample (S3) was optimized for the quantification range of dPCR according to previous reports. The ratio between the two fragments was 1.005, which closely approximated the value certified by sequencing, and the concentration was found to be 792 copies/μL. This method was precise, with an RSD of ~3%. These findings demonstrate the advantages of using the dPCR method to characterize reference materials.