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.

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.

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.

The continued use of pyrazinamide in the treatment of tuberculosis in the absence of a rapid, accurate and standardized pyrazinamide drug susceptibility assays is of great concern. While whole genome sequencing holds promise, it is not yet feasible option in low resource settings as it requires expensive instruments and bioinformatic analysis. We investigated the diagnostic performance of a closed-tube Linear-After-The-Exponential (LATE)-PCR assay for pyrazinamide susceptibility in Mycobacterium tuberculosis . Based on a set of 654 clinical Mycobacterium tuberculosis culture isolates with known mutations throughout the pncA gene as determined by Sanger sequencing, the assay displays excellent sensitivity of 96.9% (95% CI: 95.2–98.6) and specificity of 97.9% (95% CI: 96.1–99.7). In a subset of 384 isolates with phenotypic drug susceptibility testing, we also observed high sensitivity of 98.9% (95% CI: 97.5–100) but lower specificity of 91.8% (95% CI: 87.9–95.8) when compared to phenotypic drug susceptibility testing. We conclude that the LATE PCR assay offers both a rapid and accurate prediction of pyrazinamide susceptibility.

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).

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.

The present study describes a rapid, universal, easy-to-use, closed-tube, non-sequencing method that should also be able to uniquely identify almost any animal species on earth. The approach, called Virtual Barcoding, is illustrated using five species of nematodes from three genera.Linear-After-The-Exponential (LATE) PCR was used to amplify a portion of the CO1 gene for each of five commercially available, beneficial species of soil nematodes. A set of ten low temperature Lights-On/Lights-Off consensus probes were included in the reaction mixture and were used at end-point to coat the accumulated single-stranded amplicon by dropping the temperature. Because each of the probes is mis-match tolerant, the temperature at which it hybridizes to its complementary region within the target is sequence dependent. As anticipated, each species had its own unique fluorescent signature in either three different colors, or a single color depending on which fluorophores were used to label the Lights-On probes. Each fluorescent signature was then mathematically converted to a species-specific Virtual Barcode.

It is critically important to understand the molecular mechanisms of the development and progression of type 2 diabetes. One such mechanism could be mitochondrial dysfunction in organs such as the liver. It is not known if the buildup of mitochondria DNA (mtDNA) mutations (mutational load; ML) leads to dysfunction and diabetes, e.g. via insulin resistance brought about by mtDNA ML in the liver. The association between diabetes and hepatic mtDNA mutations was studied over the course of type 2 diabetes onset and progression in Nile rats, a novel model of diet‐induced diabetes that recapitulates the human disease. Age‐matched diabetic and non‐diabetic wild‐type rats were scored for disease incidence and severity using random blood glucose (RBG), hyperlipidemia, and hepatic pathology after 8‐21 weeks on diabetogenic diets after weaning. Hepatic mtDNA mutations were measured in the D‐loop and ND3 regions using digital LATE‐PCR with Lights‐On/Lights‐Off probes. Initial results showed that diabetic rats exhibited greater mutational load than the healthy controls. In separate studies mutations preceded the onset of diabetes (see intermediate rats) and palm fruit juice, an extract from the oil palm shown to delay diabetes in the Nile rat, reduced the number of mutations observed in diabetic rats (table). Mutational load did not correlate well with the severity of diabetes. Status (n) RBG (mg/dl) 30’ OGTT (mg/dl) Avg. ML Liv%bdwt Healthy 7 62 ± 9 205 ± 65 41 ± 10a, b 2.9 ± 0.3 Intermediate 7 77 ± 18 281 ± 76 70 ± 10a 2.6 ± 0.8 Diabetic 7 316 ± 214 527 ± 81 67 ± 10b 4.4 ± 0.8 Healthy 3 60 ± 30 NA 14 ± 7c, e NA Diabetic‐PFJ 3 372 ± 85 NA 67 ± 2c, d NA Diabetic+PFJ 3 239 ± 19 NA 30 ± 6c, d, e NA Intermediate Phenotype = low RBG levels, elevated oral glucose tolerance test (OGTT).Average liver weight as a percent of body mass.This research implicates that mitochondria mutagenesis plays a role in disease onset. Such mtDNA damage may serve as a potential molecular biomarker for type 2 diabetes.Supported by a grant from the Malaysian Palm Oil Board.

The amplification and detection of diverse strains of an infectious virus or bacteria, or variants within a gene family is important for both clinical and basic research but can be difficult using conventional PCR. This report describes and illustrates a novel closed-tube method for amplifying and characterizing heterogeneous target sequences using members of the CTX-M beta-lactamase gene family. Different subgroups of CTX-M genes exhibit low sequence identity, but accurate and efficient detection of these variants is critical because they all confer resistance to penicillin, cefotaxime, and other antibiotics of the beta-lactam class. The method combines a single pair of "thermodynamic consensus primers" (tcPrimers) with one or more "initiator primers" (iPrimers), added at low concentration (5-10 nM). Each iPrimer improves the initial amplification of one or more variants because it has fewer mismatches to its intended target than the more abundant tcPrimers. As a result of initial amplification, each heterogeneous sequence is shifted stepwise toward a better match with the tcPrimers. As soon as the tcPrimer hybridization takes place, amplification proceeds with high efficiency. The tcPrimer pairs can be designed for symmetric PCR or for Linear-After-The-Exponential (LATE)-PCR. LATE-PCR offers the advantage of generating single-stranded DNA that can be characterized for different gene variants in the same closed tube, using low-temperature mismatch-tolerant fluorescent 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.

Background: We have constructed a highly informative, rapid, single-tube, single-color assay for distinguishing variants in the inhA promotor, and katG and rpoB gene targets responsible for isoniazid and rifampicin resistance, using technologies invented at Brandeis University. Methods & Materials: A multiplex LATE-PCR reaction is used to generate single-stranded DNA products for the Rifampicin Resistance Determining Region of the rpoB gene, the katG gene, and the inhA promotor, as well as an amplifiable internal control. These four amplicons are detected with four sets of Therma-Light probes all of which are labeled with Black Hole Quenchers with/without Quasar 670 fluorophors. Probe-target hybridization takes place at end-point over a wide range of temperatures below the reaction annealing temperature. The temperature ranges used for the four sets of Therma-Light probes are deliberately overlapped, making it possible to maximize the amount of information generated in a single color. The amplifiable internal control generates a specific very low temperature signal and an additional non-amplifiable internal control generates a second signal at a specific high temperature. Together these controls can be used to calibrate each reaction. Another proprietary reagent is added to the reaction mixture to improve primer specificity. Results: The present assay has been tested on twenty-two different archival strains which are reported to harbor many of the common alleles responsible for resistance to rifampin and isoniazid. Each strain displayed its own “fluorescent signature” reflecting the underlying alleles present in the three target sequences. But, not every fluorescent signature agreed with archival information on the mutational composition of the strain. These discrepancies were resolved by sequencing the reaction products. The results show that the fluorescent signatures consistently identify the alleles correctly. Many more strains of M. tuberculosis will soon be tested. Conclusion: This single color multiplex assay has both clinical and research applications and is just one of many possible assays for M(X)DR-TB that can be constructed using these technologies. For instance, Therma-Light probes labeled with fluorophores in other colors can be added for simultaneous analysis variants responsible for resistance to all other first and second line antibiotics. Support: Brandeis University and Hain Lifescience

Background: In 2006, the World Health Organization (WHO) and the Stop TB partnership called for strengthening of diagnostic services and highlighted the need for the development of rapid diagnostics to fight the tuberculosis pandemic. In 2011, WHO estimated that approximately 630,000 (5.3%) of the 12 million TB cases had multiple drug resistant (MDR)-TB, while more than 80 countries have reported cases of extremely drug-resistant (XDR)-TB. Only a small fraction of reported cases (<20%) were correctly diagnosed and even fewer were treated according to WHO standards. In response the WHO endorsed the Genotype® MTBDRplus (version 1.0) line probe assay (LPA) in 2008 and the Xpert®MTB/RIF assay in 2010. But these tests only provide evidence for resistance to isoniazid and/or rifampicin. There continues to be a critical need for a more comprehensive convenient diagnostic technology. The highly multiplexed LATE-PCR assay for M(X)DR-TB described here was designed to meet that need.\ Our goal was to firmly establish that a highly multiplexed Linear-After-the-Exponential (LATE) PCR single closed-tube assay can simultaneously detect and distinguish multiple mutations in multiple gene targets that are known to confer resistance to isoniazid, rifampicin, ethambutol, ofloxacin, amikacin, kanamycin and capreomycin. Methods & Materials: In this initial study, DNA from clinical isolates with different rpoB, katG, embB, inhA promoter, gyrB, gyrA and rrs genotypes were selected from a DNA bank housed at Stellenbosch University. Each DNA samples was amplified and the singled-stranded DNA products were scanned for mutations at end-point using the same mixture of Lights-On/Lights-Off Probes. The resulting fluorescent signatures were compared to that of H37Rv, a pan-susceptible “wildtype” strain. Results: Each clinical isolate harbouring a unique mutation had its own, highly reproducible fluorescent signature distinct from that of H37Rv, as well as all other isolates with different mutations. Conclusion: This study achieved the intended transfer of the Brandeis University technology to Stellenbosch University. This study also demonstrates that this single tube multiplexed assay can simultaneously distinguish the different mutations that confer resistance to rifampicin, isoniazid, ethambutol, fluoroquinolones, aminoglycosides and ethionamide in less than three hours. (Supported by NIH Grant R01 A1099532)