Recent Advances in Closed-Tube Barcoding for FastFish-ID.

FastFish-ID for rapid and accurate identification of fish species was conceived at Brandeis University based on pioneering work on Closed-Tube Barcoding (Rice et al., Mitochondrial DNA Part A 27(2):1358-1363, 2016; Sirianni et al., Genome 59:1049-1061, 2016). FastFish-ID was subsequently validated and commercialized at Thermagenix, Inc. using a portable device and high-precision PCR (Naaum et al., Food Res Int 141:110035, 2021). The motivation for these efforts was the pressing need for a technology that could be widely used throughout the seafood supply chain to combat IUU Fishing (Helyar et al., PLOS ONE 9, 2014) and overfishing (FAO, State of the World Fisheries and Aquaculture 2018. http://www.fao.org/documents/card/en/c/I9540EN/ , 2018), along with seafood fraud and mislabeling (Watson et al., Fish Fish 17:585-595, 2015). These destructive practices are wasting fish stocks, frustrating attempts to achieve seafood sustainability, endangering oceanic ecosystems, and causing consumers billions of dollars each year (Porterfield et al., Oceana: February, 2022). During the past three Covid19 pandemic years, EcologeniX, LLC has taken over further development and optimization of FastFish-ID. The present chapter provides an overview of the improvements introduced throughout the FastFish-ID process.

Recent Applications of FastFish-ID.

FastFish-ID via Closed-Tube barcoding is a portable platform for rapid and accurate identification of fish species that was conceived at Brandeis University, commercialized at Thermagenix, Inc., and further improved at Ecologenix, LLC (see Chap. 17 in this volume). This chapter focuses on the use of FastFish-ID for (1) identification of intraspecies variants, (2) quantitative use of FastFish-ID to measure the decay of fresh fish, and (3) use of FastFish-ID for the identification of dried and processed shark fins.

Validation of FASTFISH-ID: A new commercial platform for rapid fish species authentication via universal closed-tube barcoding.

Seafood represents up to 20% of animal protein consumption in global food consumption and is a critical dietary and income resource for the world's population. Currently, over 30% of marine fish stocks are harvested at unsustainable levels, and the industry faces challenges related to Illegal, Unregulated and Unreported (IUU) fishing. Accurate species identification is one critical component of successful stock management and helps combat fraud. Existing DNA-based technologies permit identification of seafood even when morphological features are removed, but are either too time-consuming, too expensive, or too specific for widespread use throughout the seafood supply chain. FASTFISH-ID is an innovative commercial platform for fish species authentication, employing closed-tube barcoding in a portable device. This method begins with asymmetric PCR amplification of the full length DNA barcode sequence and subsequently interrogates the resulting single-stranded DNA with a universal set of Positive/Negative probes labeled in two fluorescent colors. Each closed-tube reaction generates two species-specific fluorescent signatures that are then compared to a cloud-based library of previously validated fluorescent signatures. This novel approach results in rapid, automated species authentication without the need for complex, time consuming, identification by DNA sequencing, or repeated analysis with a panel of species-specific tests. Performance of the FASTFISH-ID platform was assessed in a blinded study carried out in three laboratories located in the UK and North America. The method exhibited a 98% success rate among the participating laboratories when compared to species identification via conventional DNA barcoding by sequencing. Thus, FASTFISH-ID is a promising new platform for combating seafood fraud across the global seafood supply chain.

Diagnostic Accuracy and Utility of FluoroType MTBDR, a New Molecular Assay for Multidrug-Resistant Tuberculosis.

Most cases of multidrug-resistant (MDR) tuberculosis (TB) are never diagnosed (328,300 of the ∼490,000 cases in 2016 were missed). The Xpert MTB/RIF assay detects resistance only to rifampin, despite ∼20% of rifampin-resistant cases being susceptible to isoniazid (a critical first-line drug). Consequently, many countries require further testing with the GenoType MTBDRplus assay. However, MTBDRplus is not recommended for use on smear-negative specimens, and thus, many specimens require culture-based drug susceptibility testing. Furthermore, MTBDRplus requires specialized expertise, lengthy hands-on time, and significant laboratory infrastructure and interpretation is not automated. To address these gaps, we evaluated the accuracy of the FluoroType MTBDR (FluoroType) assay. Sputa from 244 smear-positive and 204 smear-negative patients with presumptive TB (Xpert MTB positive, n = 343) were tested. Culture and MTBDRplus on isolates served as reference standards (for active TB and MDR-TB, respectively). Sanger sequencing and MTBDRplus, both of which were performed on sputa, were used to resolve discrepancies. The sensitivity of FluoroType for the detection of M. tuberculosis complex was 98% (95% confidence interval [CI], 95 to 99%) and 92% (95% CI, 84 to 96%) for smear-positive and smear-negative specimens, respectively (232/237 versus 90/98 specimens; P < 0.009). The sensitivity and specificity for smear-negative specimens were 100% and 97%, respectively, for rifampin resistance; 100% and 98%, respectively, for isoniazid resistance; and 100% and 100%, respectively, for MDR-TB. FluoroType identified 98%, 97%, and 97% of the rpoB, katG, and inhA promoter mutations, respectively. FluoroType has excellent sensitivity with sputa equivalent to that of MTBDRplus with the isolates and can provide rapid drug susceptibility testing for rifampin and isoniazid. In addition, the capacity of FluoroType to simultaneously identify virtually all mutations in the rpoB, katG, and inhA promoter may be useful for individualized treatment regimens.

Closed-Tube Barcoding.

Here, we present a new approach for increasing the rate and lowering the cost of identifying, cataloging, and monitoring global biodiversity. These advances, which we call Closed-Tube Barcoding, are one application of a suite of proven PCR-based technologies invented in our laboratory. Closed-Tube Barcoding builds on and aims to enhance the profoundly important efforts of the International Barcode of Life initiative. Closed-Tube Barcoding promises to be particularly useful when large numbers of small or rare specimens need to be screened and characterized at an affordable price. This approach is also well suited for automation and for use in portable devices.

Applying X-ray Imaging Crystal Spectroscopy for Use as a High Temperature Plasma Diagnostic.

X-ray spectra provide a wealth of information on high temperature plasmas; for example electron temperature and density can be inferred from line intensity ratios. By using a Johann spectrometer viewing the plasma, it is possible to construct profiles of plasma parameters such as density, temperature, and velocity with good spatial and time resolution. However, benchmarking atomic code modeling of X-ray spectra obtained from well-diagnosed laboratory plasmas is important to justify use of such spectra to determine plasma parameters when other independent diagnostics are not available. This manuscript presents the operation of the High Resolution X-ray Crystal Imaging Spectrometer with Spatial Resolution (HIREXSR), a high wavelength resolution spatially imaging X-ray spectrometer used to view hydrogen- and helium-like ions of medium atomic number elements in a tokamak plasma. In addition, this manuscript covers a laser blow-off system that can introduce such ions to the plasma with precise timing to allow for perturbative studies of transport in the plasma.

Single-Tube Mutation Scanning of The Epidermal Growth Factor Receptor Gene Using Multiplex LATE-PCR and Lights-On/Lights-Off Probes.

BACKGROUND: Numerous mutations in exons 18-21 of the epidermal growth factor receptor (EGFR) gene determine the response of many patients with non-small cell lung carcinoma (NSCLC) to anti-EGFR tyrosine kinase inhibitors (TKIs). This paper describes a single closed-tube assay for simultaneous mutational scanning of EGFR exons 18-21. METHODS: The assay first co-amplifies all four exons as separate single-stranded DNA products using Linear-After-The-Exponential (LATE)-PCR. The amplicons are then interrogated at endpoint along their length using sets of Lights-On/Lights-Off probes of a different color for each exon. The four resulting fluorescent signatures are unique for each underlying DNA sequence. Every mutation in a target potentially alters its unique fluorescent signature thereby revealing the presence of the mutation. RESULTS: The assay readily detects mutations which cause sensitivity or resistance to TKIs and can distinguish these clinically important genetic changes from silent mutations which have no impact on protein function. The assay identifies as little as 5% mutant sequences in mixtures of normal DNA and mutant DNA prepared from cancer cell lines. Proof-of-principle experiments demonstrate mutation identification in formalin-fixed, paraffin-embedded NSCLC biopsies. CONCLUSION: The LATE-PCR EGFR assay described here represents a new type of highly informative, single-tube diagnostic test for mutational scanning of multiple gene coding regions and/or multiple gene targets for personalized cancer therapies.

Rapid detection of TEM-type extended-spectrum ß-lactamase (ESBL) mutations using lights-on/lights-off probes with single-stranded DNA amplification.

Rapid identification of specific TEM-type ß-lactamase genes in bacterial infections is important for determining appropriate clinical treatment. We report here the design and initial testing of a molecular diagnostic assay capable of amplifying a large segment of the blaTEM gene, as well as detecting widely spaced extended-spectrum ß-lactamase (ESBL) mutations and inhibitor-resistant TEM (IRT) mutations (eg, clavulanic acid resistance). Single-stranded DNA is generated using linear-after-the-exponential PCR (LATE-PCR) and is analyzed at the endpoint, using a set of four fluorescently labeled and four quencher-labeled probes in a single closed tube. These lights-on/lights-off probes work in concert to generate sequence-specific fluorescence contours over a temperature range from 25°C to 75°C. Mutant sequences from synthetic TEM gene variants and from TEM gene variants in bacterial strains generated large increases in fluorescent signal relative to that from the reference sequence for TEM-1. Clinical use of this convenient, single-closed-tube assay would make it possible to rapidly distinguish ESBL from non-ESBL variants and thereby to begin early treatment with suitable antibiotics.

Fluorescent signatures for variable DNA sequences.

Life abounds with genetic variations writ in sequences that are often only a few hundred nucleotides long. Rapid detection of these variations for identification of genetic diseases, pathogens and organisms has become the mainstay of molecular science and medicine. This report describes a new, highly informative closed-tube polymerase chain reaction (PCR) strategy for analysis of both known and unknown sequence variations. It combines efficient quantitative amplification of single-stranded DNA targets through LATE-PCR with sets of Lights-On/Lights-Off probes that hybridize to their target sequences over a broad temperature range. Contiguous pairs of Lights-On/Lights-Off probes of the same fluorescent color are used to scan hundreds of nucleotides for the presence of mutations. Sets of probes in different colors can be combined in the same tube to analyze even longer single-stranded targets. Each set of hybridized Lights-On/Lights-Off probes generates a composite fluorescent contour, which is mathematically converted to a sequence-specific fluorescent signature. The versatility and broad utility of this new technology is illustrated in this report by characterization of variant sequences in three different DNA targets: the rpoB gene of Mycobacterium tuberculosis, a sequence in the mitochondrial cytochrome C oxidase subunit 1 gene of nematodes and the V3 hypervariable region of the bacterial 16 s ribosomal RNA gene. We anticipate widespread use of these technologies for diagnostics, species identification and basic research.

Dilute-'N'-Go dideoxy sequencing of all DNA strands generated in multiplex LATE-PCR assays.

We have recently described a Dilute-'N'-Go protocol that greatly simplifies preparation and sequencing of both strands of an amplicon generated using linear-after-the-exponential (LATE)-PCR, an advanced form of asymmetric PCR . The same protocol can also be used to sequence all limiting primer strands in a multiplex LATE-PCR, by adding back each of the depleted limiting primers to a separate aliquot of the multiplex reaction. But, Dilute-'N'-Go sequencing cannot be used directly to sequence each of the excess primer strands in the same multiplex reaction, because all of the excess primers are still present at high concentration. This report demonstrates for the first time that it is possible to sequence each of the excess primer strands using a modified Dilute-'N'-Go protocol in which blockers are added to prevent all but one of the excess primers serving as the sequencing primer in separate aliquots. The optimal melting temperatures, positions and concentrations of blockers relative to their corresponding excess primers are defined in detail. We are using these technologies to measure DNA sequence changes in mitochondrial genomes that accompany aging and exposure to certain drugs.

Monoplex/multiplex linear-after-the-exponential-PCR assays combined with PrimeSafe and Dilute-'N'-Go sequencing.

This protocol describes the design and execution of monoplex and multiplex linear-after-the-exponential (LATE)-PCR assays using a novel reagent, PrimeSafe, that suppresses all forms of mispriming. LATE-PCR is an advanced form of asymmetric amplification that uses a limiting primer and an excess primer for efficient exponential amplification of double-stranded DNA, followed by linear amplification of one strand. Each single-stranded amplicon can be quantitatively detected in real time or at end point. By separating primer annealing from product detection, LATE-PCR enables product analysis at low temperatures. Alternatively, each single strand can be sequenced by a convenient Dilute-'N'-Go procedure. Amplified samples are diluted with individual sequencing primers without the use of columns or spins. We have amplified and then sequenced 15 different single-stranded products generated in a single multiplexed LATE-PCR comprised of 15 pairs of unrelated primers. Dilute-'N'-Go dideoxy sequencing is more convenient, faster and less expensive than sequencing double-stranded amplicons generated via conventional symmetric PCR. The preparation of LATE-PCR products for Dilute-'N'-Go sequencing takes only 30 seconds.

Two-temperature LATE-PCR endpoint genotyping.

BACKGROUND: In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay. RESULTS: Two-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE)-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA) and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of 2:1 and 1:2 ratios of the same alleles. CONCLUSION: SNP genotyping via Two-Temperature LATE-PCR takes place in a homogeneous closed-tube format and uses a single hybridization probe per SNP site. These assays are convenient, rely on endpoint analysis, improve the options for construction of multiplex assays, and are suitable for SNP genotyping, mutation scanning, and detection of DNA duplication or deletions.

Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)-PCR.

Pyrosequencing is a highly effective method for quantitatively genotyping short genetic sequences, but it currently is hampered by a labor-intensive sample preparation process designed to isolate single-stranded DNA from double-stranded products generated by conventional PCR. Here linear-after-the-exponential (LATE)-PCR is introduced as an efficient and potentially automatable method of directly amplifying single-stranded DNA for pyrosequencing, thereby eliminating the need for solid-phase sample preparation and reducing the risk of laboratory contamination. These improvements are illustrated for single-nucleotide polymorphism genotyping applications, including an integrated single-cell-through-sequencing assay to detect a mutation at the globin IVS 110 site that frequently is responsible for beta-thalassemia.

Linear-After-The-Exponential (LATE)-PCR: primer design criteria for high yields of specific single-stranded DNA and improved real-time detection.

Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations. The present report systematically examines the primer design parameters that affect the exponential and linear phases of LATE-PCR amplification. In particular, we investigated how altering the concentration-adjusted melting temperature (Tm) of the limiting primer (TmL) relative to that of the excess primer (TmX) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when TmL - TmX 5 degrees C. We also investigated how altering TmX relative to the higher Tm of the double-stranded amplicon (TmA) affects the rate and extent of linear amplification. Excess primers with TmX closer to TmA yielded higher rates of linear amplification and stronger signals from a hybridization probe. These design criteria maximize the yield of specific single-stranded DNA products and make LATE-PCR more robust and easier to implement. The conclusions were validated by using primer pairs that amplify sequences within the cystic fibrosis transmembrane regulator (CFTR) gene, mutations of which are responsible for cystic fibrosis.

Linear-after-the-exponential (LATE)-PCR: an advanced method of asymmetric PCR and its uses in quantitative real-time analysis.

Conventional asymmetric PCR is inefficient and difficult to optimize because limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature. Linear-After-The-Exponential (LATE)-PCR describes a new paradigm for primer design that renders assays as efficient as symmetric PCR assays, regardless of primer ratio. LATE-PCR generates single-stranded products with predictable kinetics for many cycles beyond the exponential phase. LATE-PCR also introduces new probe design criteria that uncouple hybridization probe detection from primer annealing and extension, increase probe reliability, improve allele discrimination, and increase signal strength by 80-250% relative to symmetric PCR. These improvements in PCR are particularly useful for real-time quantitative analysis of target numbers in small samples. LATE-PCR is adaptable to high throughput applications in fields such as clinical diagnostics, biodefense, forensics, and DNA sequencing. We showcase LATE-PCR via amplification of the cystic fibrosis CFDelta508 allele and the Tay-Sachs disease TSD 1278 allele from single heterozygous cells.

Detection of cystic fibrosis alleles from single cells using molecular beacons and a novel method of asymmetric real-time PCR.

We present a method for rapid and accurate identification of the normal and DeltaF508 alleles of the cystic fibrosis (CF) gene in single human cells that utilizes LATE (linear after the exponential)-PCR, a newly invented form of asymmetric PCR. Detection of the single-stranded amplicon is carried out in real time, using allele-specific molecular beacons. The LATE-PCR method permits controlled abrupt transition from exponential to linear amplification and thereby enhances the fluorescent signals and reduces variability between replicate samples relative to those obtained using typical real-time PCR. Of 239 single lymphoblasts generating amplification signals, 227 (95%) exhibited signals that met objective quantitative criteria required for diagnosis. Among these samples, 222 were genotyped correctly, for an assay accuracy of 98%. The small number of diagnostic errors was due to allele drop-out among heterozygous lymphoblasts, 4/119 (3.4%), and contamination among homozygous DeltaF508 lymphoblasts, 1/57 (1.8%). LATE-PCR offers a new strategy for preimplantation genetic diagnosis and other fields in which accurate quantitative detection of single copy genes is important.

Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.

Xist gene expression begins at the late 2-cell stage in female mouse embryos and by the third division results in the accumulation of an average 100 copies of Xist RNA per cell, as measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). In the blastocyst, the trophectoderm maintains the paternally imprinted pattern of Xist expression present during early development, while either the maternal or the paternal X chromosome can express Xist among cells of the inner mass. Fluorescent in situ hybridization (FISH) has previously established that Xist transcripts are localized on the silenced X chromosome, forming aggregates of variable dimensions in blastomeres of 8-cell embryos. This observation and the fact that Xist RNA accumulation per cell sharply decreases after morula stage raise the possibility that cells of cleaving embryos contain different levels of Xist RNA, perhaps linked to their subsequent developmental fates. We show here that Xist RNA is efficiently recovered from single blastomeres isolated from 8-cell embryos following laser zona drilling. Sexing of the samples and simultaneous quantification of Xist RNA in individual cells is achieved with a multiplex Xist/Sry real-time RT-PCR assay sensitive to the single-copy level. This analysis reveals that Xist RNA is indeed accumulated to substantially different levels in individual blastomeres of the same 8-cell embryo and that two blastomeres contain most of the molecules per embryo. These results support the conclusion that cells of the early mammalian embryo are not all functionally equivalent. Differential Xist gene expression could arise from differences in DNA methylation, or the order in which cells divide.

Real-time PCR with molecular beacons provides a highly accurate assay for detection of Tay-Sachs alleles in single cells.

The results presented here provide the first single-cell genetic assay for Tay-Sachs disease based on real-time PCR. Individual lymphoblasts were lysed with an optimized lysis buffer and assayed using one pair of primers that amplifies both the wild type and 1278 + TATC Tay-Sachs alleles. The resulting amplicons were detected in real time with two molecular beacons each with a different colored fluorochrome. The kinetics of amplicon accumulation generate objective criteria by which to evaluate the validity of each reaction. The assay had an overall utility of 95%, based on the detection of at least one signal in 235 of the 248 attempted tests and an efficiency of 97%, as 7 of the 235 samples were excluded from further analysis for objective quantitative reasons. The accuracy of the assay was 99.1%, because 228 of 230 samples gave signals consistent with the genotype of the cells. Only two of the 135 heterozygous samples were allele drop-outs, a rate far lower than previously reported for single-cell Tay-Sachs assays using conventional methods of PCR.

QuantiLyse: reliable DNA amplification from single cells.

Amplification of DNA sequencesfrom single cells via PCR is increasingly used in basic research and clinical diagnostics but remains technically difficult. We have developed a cell lysis protocol that uses an optimized proteinase K solution, named QuantiLyse and permits reliable amplification from individual cells. This protocol was compared to other published methods by means of real-time PCR with molecular beacons. The results demonstrate that QuantiLyse treatment of single lymphocytes renders gene targets more availablefor amplification than other published proteinase K methods or lysis in water. QuantiLyse and an optimized alkaline lysis were equally effective in terms of target availability, although QuantiLyse offers greaterflexibility, as it does not require neutralization and can comprise a higher percentage of the final PCR volume. Maximum gene target availability is also obtained following QuantiLyse treatment of samples containing up to 10000 cells (the largest number tested). Thus, QuantiLyse maximizes the chances that targeted DNA sequences will be available for amplification during the first cycle of PCR, thereby reducing the variability among replicate reactions as well as the likelihood of amplification failure or allele drop-out. QuantiLyse will be useful in a range of investigations aimed at gene detection in small numbers of cells.

Developmentally-regulated changes of Xist RNA levels in single preimplantation mouse embryos, as revealed by quantitative real-time PCR.

Xist RNA localizes to the inactive X chromosome in cells of late cleavage stage female mouse embryos (Sheardown et al., 1997: Cell 91:99-107). Fluorescence in situ hybridization (FISH), however, does not quantify the number of Xist transcripts per nucleus. We have used real-time reverse transcription-polymerase chain reaction (RT-PCR) to measure Xist RNA levels in single preimplantation embryos and to establish developmental profiles in both female and male samples. The gender of each embryo was readily established based on Xist RNA levels, by counting Xist gene copies per cell, and by independent detection of the presence/absence of Sry, a Y chromosome-specific gene. Xist expression in males was found to be very low at all stages, as suggested by FISH. In contrast, female embryos contained measurable levels of Xist mRNA starting at the late 2-cell stage and rapidly accumulated Xist transcripts until morula stage. Xist RNA accumulation per embryo then reached a plateau, while cell division continued. We propose that during early cleavage high enough levels of Xist mRNA are transcribed to generate a pool of unbound molecules. This pool would serve to temporarily maintain X chromosome inactivation without additional transcription while the trophectoderm and inner cell mass (ICM) differentiate. The ICM would then loose the paternally imprinted pattern of X inactivation originally present in all embryonic cells.