Assay For Detection Of DNA Research Articles

A human papillomavirus whole genome plasmid repository: A resource for HPV DNA quality control reagents.

Well characterized reference reagents are useful for assay validation, proficiency/competency assessment, daily run controls, and to improve inter-laboratory comparisons. Synthetic human papillomavirus (HPV) DNA fragments and plasmid clones are available, but synthetic fragments include limited segments of the HPV genome and many HPV plasmids have interrupted coding regions or contain partial genomes. As a result, they are not compatible with all HPV DNA detection and typing assays. To address this need, we are establishing an HPV plasmid repository of HPV clones containing the whole genome of each type with no interruptions in coding regions. To date, HPV plasmid clones for 16 HPV types, (including all vaccine types and 14 types in clinical assays: HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) have been constructed using a Gibson assembly method and validated by sequencing and the Novaplex HPV typing assay. The newly constructed HPV whole genome plasmids can serve as a quality control reagent resource for HPV DNA assays and are available for public health and research laboratories.

Duplex loop-mediated isothermal amplification assay for simultaneous detection of human and human male DNA

ObjectiveScreening of human and human male DNA is necessary for forensic DNA analyses. Although quantitative real-time PCR (qPCR) is commonly used for detecting and quantifying these DNA targets, its use as a screening tool is time-consuming and labor-intensive. To streamline and simplify the screening process, we aimed to develop a duplex loop-mediated isothermal amplification (LAMP) assay capable of simultaneously detecting human and human male DNA in a single tube. We assessed the duplex LAMP assay for forensic application.ResultsFor our duplex LAMP assay, we have utilized two fluorescent probes with HEX and FAM fluorophores to specifically detect human and human male DNA, respectively. The HEX (human target) signal was detected from both the male and female DNA samples, and the FAM (male target) signal was detected from only the male DNA sample. This assay has a sensitivity of 10–1 pg of DNA for both targets. Additionally, we successfully detected the two targets in the DNA samples extracted from forensically relevant body fluids, including blood, saliva, semen, and vaginal secretions.

Type distribution of human papillomaviruses in ThinPrep cytology samples and HPV16/18 E6 gene variations in FFPE cervical cancer specimens in Fars province, Iran

BackgroundThere exists strong evidence that human papillomavirus (HPV) is associated with cervical cancer (CC). HPV E6 is a major oncogene whose sequence variations may be associated with the development of CC. There is not sufficient data on the distribution of HPV types in ThinPrep cytology specimens and HPV 16/18 E6 gene variations among CC patients in the southwest of Iran. This study was conducted to contribute to HPV screening and vaccination in Iran.MethodsA total of 648 women screened for cervicitis, intraepithelial neoplasia or CC were included in the study. All participants underwent ThinPrep cytology testing, single-step HPV DNA detection and allele-specific reverse hybridization assays. Moreover, a total of 96 specimens previously tested positive for single infection with HPV16 or 18 were included for variant analysis. HPV16/18 lineages and sublineages were determined by PCR assays followed by sequencing the E6 gene and the construction of neighbor-joining phylogenetic trees.ResultsOverall, HPV DNA was detected in 62.19% of all the screened subjects. The detection rates of HPV DNA among individuals with normal, ASC-US, ASC-H, LSIL, and HSIL cervical cytology were 48.9%, 93.6%, 100%, 100%, and 100%, respectively. Low-risk HPVs were detected more frequently (46.9%) than high-risk (38.9%) and possible high-risk types (11.1%). Of 403 HPV-positive subjects, 172 (42.7%) had single HPV infections while the remaining 231 (57.3%) were infected with multiple types of HPV. Our results indicated a remarkable growth of high-risk HPV66 and 68 and low-risk HPV81 which have rarely been reported in Iran and HPV90 and 87 that are reported for the first time in the country. In addition, 3 lineages (A, D, and C) and 6 sublineages (A1, A2, A4, C1, D1, and D2) of HPV16, and one lineage and 4 sublineages (A1, A3, A4, and A5) of HPV18 were identified. The studied HPV16 and 18 variants mainly belonged to the D1 and A4 sublineages, respectively.ConclusionThe present study suggests that the prevalence of HPV infection in women of all age groups with or without premalignant lesions in the southwestern Iran is high and the predominant HPV types in the southwest of Iran may differ from those detected in other parts of the country. This study also highlights the necessity of not only initiating HPV vaccination for the general population but also developing new vaccines that confer immunity against the prevalent HPV types in the area and national cervical screening programs using a combination of thinPrep cytology test and HPV detection assays in order to improve the accuracy of the screening.

(356) A Novel Multiplex Digital PCR Assay for Same-Day Detection of Donor-Derived Cell-Free DNA in Pediatric Heart Transplant Recipients

(356) A Novel Multiplex Digital PCR Assay for Same-Day Detection of Donor-Derived Cell-Free DNA in Pediatric Heart Transplant Recipients

Mycobacterium tuberculosis complex detection in rural goat herds in South Africa using Bayesian latent class analysis

Animal tuberculosis affects a wide range of domestic and wild animal species, including goats (Capra hircus). In South Africa, Mycobacterium tuberculosis complex (MTBC) testing and surveillance in domestic goats is not widely applied, potentially leading to under recognition of goats as a potential source of M. bovis spread to cattle as well as humans and wildlife. The aim of this study was to estimate diagnostic test performance for four assays and determine whether M. bovis infection was present in goats sharing communal pastures with M. bovis positive cattle in the Umkhanyakude district of Northern Zululand, KwaZulu Natal. In 2019, 137 M. bovis-exposed goats were screened for MTBC infection with four diagnostic tests: the in vivo single intradermal comparative cervical tuberculin test (SICCT), in vitro QuantiFERON®-TB Gold (QFT) bovine interferon-gamma release assay (IGRA), QFT bovine interferon gamma induced protein 10 (IP-10) release assay (IPRA), and nasal swabs tested with the Cepheid GeneXpert® MTB/RIF Ultra (GXU) assay for detection of MTBC DNA. A Bayesian latent class analysis was used to estimate MTBC prevalence and diagnostic test sensitivity and specificity. Among the 137 M. bovis-exposed goats, positive test results were identified in 15/136 (11.0%) goats by the SICCT; 4/128 (3.1%) goats by the IPRA; 2/128 (1.6%) goats by the IGRA; and 26/134 (19.4%) nasal swabs by the GXU. True prevalence was estimated by our model to be 1.1%, suggesting that goats in these communal herds are infected with MTBC at a low level. Estimated posterior means across the four evaluated assays ranged from 62.7% to 80.9% for diagnostic sensitivity and from 82.9% to 97.9% for diagnostic specificity, albeit estimates of the former (diagnostic sensitivity) were dependent on model assumptions. The application of a Bayesian latent class analysis and multiple ante-mortem test results may improve detection of MTBC, especially when prevalence is low. Our results provide a foundation for further investigation to confirm infection in communal goat herds and identify previously unrecognized sources of intra- and inter-species transmission of MTBC.

Enhancing single-molecule sensing and superresolution with DNA nanotech.

In recent years, DNA nanotechnology has matured to enable robust production of complex nanostructures and hybrid materials. We have combined DNA nanotechnology with sensitive optical detection to create functional single-molecule devices that enable new applications in single-molecule biophysics, biosensing and superresolution. To improve the sensitivity of DNA detection assays, we created DNA origami optical antennas for metal enhanced fluorescence. Two gold nanoparticles or nanorods enhance the light-matter interactions of dyes and biomolecular assays placed between them. We first discuss the influence of the metal nanostructure on the photophysical properties of dyes. We then show how single molecules can even be detected on a battery-driven portable smartphone microscope using DNA origami nanoantennas [1]. We further present our work on other biosensing applications of DNA origami as well as the possibility of isotropic nanometric superresolution by combining graphene energy transfer with pMINFLUX and DNA PAINT [2].

Validation of a Real-Time PCR Assay for Identification of Fresh and Processed Carica papaya Botanical Material: Using Synthetic DNA to Supplement Specificity Evaluation.

Several commercially important botanicals have a lack of diagnostic testing options that can quickly and unambiguously identify materials of different matrices. Real-time PCR can be a useful, orthogonal approach to identification for its exceptional specificity and sensitivity. Carica papaya L. is a species with a lack of available identification methods, and one which features two distinct commercially relevant matrices: fresh fruit and powdered fruit extract. In this study, we demonstrate the successful design and validation of a real-time PCR assay for detection of papaya DNA extracted from the two matrices. We also propose a technique that can be used during exclusivity panel construction, when genuine botanical samples are not available for certain species: substitution with synthetic DNA. We demonstrate the use of this material to complete a comprehensive specificity evaluation and confidently determine suitable Ct cutoff values. Further, we demonstrate how ddPCR can be used to determine the copy number of the target sequence in a set amount of genomic DNA, to which synthetic DNA samples can be corrected, and how it can verify specificity of the primers and probe. Through the presentation of successful assay validation for papaya detection, this work serves as a guideline for how to approach specificity evaluation when non-target botanical samples are difficult to obtain and otherwise may not have been included in the exclusivity panel.

A facile enzyme-assisted multiple recycling amplification strategy for ultrasensitive fluorescence detection of HIV-1 DNA

We developed an enzyme-assisted multiple signal amplification reaction for target nucleic acid detection. In this strategy, the target nucleic acid as triggers, and in the presence of two specific primers, DNA polymerase and DNA nicking endonuclease act in concert to promote the repetitive cycle DNA replication process, leading to an exponential increase in nucleic acid sequence. Both ends of each sequence of the amplification products and the specific primer could from a unit of Mg2+-DNAzyme which in the presence of cofactor Mg2+ could recognize and cyclically cleave the hairpin probes in the solution and thus realize real-time fluorescence detection of HIV-1 DNA. Under optimal conditions, the detection limit of this method is 10−17 M. The simple in design, isothermal conditions and easy fluorescence measurement of this method exhibits superior sensitivity and specificity, and thus holds great potential to be a promising assay for quantitative detection of DNA or RNA in theoretical and clinic studies.

Loop-mediated isothermal amplification assay for fluorescence analysis and lateral flow detection of male DNA

Male DNA screening is important in forensic investigations, such as sexual assault cases. Although quantitative real-time PCR is a robust method for detection of male DNA, it is time-consuming and labor-intensive. We herein report the development of a male DNA-targeted loop-mediated isothermal amplification (LAMP) assay that can be used for both laboratory-based fluorescence analysis and on-site lateral flow detection. The two detection systems are independent, but we streamlined the reaction before the detection by introducing a fluorescence probe and biotin-labeled primer into a single reaction. This allowed the evaluation of fluorescence signal followed by lateral flow detection. Both the fluorescence and lateral flow analyses detected as low as 10 pg of male DNA. We also integrated an alkaline lysis method with our LAMP assay. The direct assay successfully detected male DNA from forensic samples without purification. The workflow requires only <40 min for fluorescence analysis and <45 min for lateral flow detection. Furthermore, when combined with a lateral flow strip, this workflow does not require any sophisticated instruments. These findings suggest that our assay is a promising strategy for on-site male DNA screening as well as laboratory-based testing.

Abstract P2-01-15: Developing highly sensitive high NGS data efficient ctDNA detection assays for breast cancer surveillance

Abstract Introduction: Growing data established the importance of monitoring dynamic changes in circulating tumor DNA (ctDNA) to identify early signs of therapeutic responses, allowing for timely management of treatment to achieve more effective personalized therapy. Higher assay accuracy and consistency, and lower assay cost will support more clinical validation trials and benefit more cancer patients with non-invasive ctDNA NGS tests that can simultaneously map multiple genomic alterations at an affordable price. Method: The NVIGEN X - Precision Cancer Profiling test is a next generation sequencing (NGS) based circulating tumor DNA detection assay using the hybridization capture approach with customized gene panels. Our ctDNA NGS assay was developed with the use of high performance magnetic nanobeads, which enhances assay workflow at key steps including cfDNA extraction, NGS library preparation, and target enrichment. Experiments with individual plasma samples and DNA mutant fragments spiked in plasma samples were carried out to establish the assay performance such as sensitivity, specificity, consistency and data efficiency. NGS data QC metrics of the NVIGEN assay were compared with other assays in peer reviewed publications. Results: We developed a focus 32 gene panel that covers 144 kb of gene regions of clinical significance for breast cancer treatment monitoring and guidance, such as AKT1, ERBB2, PIK3CA, EGFR, ESR1, BRCA1/2, and CD274. Our results demonstrated the capability of NVIGEN X ctDNA NGS assay to detect rare copies (8 cp) of gene mutation at 0.07% MAF from DNA mutant fragments spiked into plasma samples. The NVIGEN X ctDNA NGS assays consistently presented 2-5% duplication rate, &amp;gt;80% on-target rate, &amp;lt;10% CV for key NGS data metrics, and on average required 1.36X paired reads per 1X unique coverage. Compared with the Roche Avenio assays (targeted, expanded and surveillance panels) as published in 2020 which on average required 9.36X paired reads per 1X unique coverage, the NVIGEN X -precision cancer profiling assays demonstrated 85% reduction in NGS data need to generate each unique coverage. Compared with the original Capp-seq data as published in the 2014 Nature Medicine paper which required in average 13.78 or 27.56 paired reads per unique coverage, the NVIGEN X assay demonstrated &amp;gt;90% reduction in NGS data need per unique coverage. Conclusion: The NVIGEN X - Precision Cancer Profiling assay provided high NGS assay performance with high sensitivity, specificity, and consistency, and significantly improved NGS data efficiency. This allows for dramatically reduced assay cost and will help support routine applications of ctDNA NGS tests to improve cancer patient treatment. Experiments of applying NVIGEN X assays for clinical research with patient samples are ongoing and will be presented. Citation Format: Aihua Fu, Wenwu Cui, Minh V. Ton, Kevan Wang, Weiwei Gu, Tianhong Li, Heather A. Parsons, Minetta C. Liu, George W. Sledge. Developing highly sensitive high NGS data efficient ctDNA detection assays for breast cancer surveillance [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-15.

Development of environmental DNA detection assays for snakes in paddy fields in Japan

Development of environmental DNA detection assays for snakes in paddy fields in Japan

A Model System for Sensitive Detection of Viable E. coli Bacteria Combining Direct Viability PCR and a Novel Microarray-Based Detection Approach

We established an innovative approach that included direct, viability, and nested PCR for rapid and reliable identification of the fecal indicator organism Escherichia coli (E. coli). Direct PCR enabled successful amplification of the target uidA gene, omitting a prior DNA isolation or purification step. Furthermore, we applied viability PCR (v-PCR) to ensure the detection of only relevant viable bacterial cells. The principle involves the binding of propidium monoazide (PMA), a selective nucleic acid intercalating dye, to accessible DNA of heat killed bacteria cells and, consequently, allows viable and heat killed E. coli cells to be discriminated. To ensure high sensitivity, direct v-PCR was followed by a nested PCR step. The resulting amplicons were analyzed by a rapid 30 min microarray-based DNA hybridization assay for species-specific DNA detection of E. coli. A positive signal was indicated by enzymatically generated silver nanoparticle deposits, which served as robust endpoint signals allowing an immediate visual readout. The presented novel protocol allows the detection of 1 × 101 viable E. coli cells per PCR run.

Development of gold nanoparticle-based visual assay for rapid detection of Escherichia coli specific DNA in milk of cows affected with mastitis

For early diagnosis of infections, a rapid, sensitive and precise method to identify microbial DNA coming from veterinary clinical samples, such as milk of cows affected with mastitis, is fascinating. We developed an amplification-free visual assay for rapid and sensitive detection of specific DNA from Escherichia coli, particularly uid A gene encoding for beta-glucuronidase, isolated in milk of cows affected with mastitis, based on multiple gold nanoparticles (AuNPs) captured on a magnetic microbead surface, leading to plasmonic signal enhancement, thereby improving overall sensitivity of the assay. This test can be performed in 1–1.5 h after post template DNA preparation and can be visualized with naked eyes without the use of any expensive equipment. The visual assay detects a minimum microbial load of 102 CFU/μl and can identify bacterial DNA as low as 1 pg. This test provides a precise detection tool for E coli in clinical samples, such as milk of cows affected with mastitis, as a quick and user-friendly molecular detection approach.

Development of an Automated, Non-Enzymatic Nucleic Acid Amplification Test.

Among nucleic acid diagnostic strategies, non-enzymatic tests are the most promising for application at the point of care in low-resource settings. They remain relatively under-utilized, however, due to inadequate sensitivity. Inspired by a recent demonstration of a highly-sensitive dumbbell DNA amplification strategy, we developed an automated, self-contained assay for detection of target DNA. In this new diagnostic platform, called the automated Pi-powered looping oligonucleotide transporter, magnetic beads capture the target DNA and are then loaded into a microfluidic reaction cassette along with the other reaction solutions. A stepper motor controls the motion of the cassette relative to an external magnetic field, which moves the magnetic beads through the reaction solutions automatically. Real-time fluorescence is used to measure the accumulation of dumbbells on the magnetic bead surface. Left-handed DNA dumbbells produce a distinct signal which reflects the level of non-specific amplification, acting as an internal control. The autoPiLOT assay detected as little as 5 fM target DNA, and was also successfully applied to the detection of S. mansoni DNA. The autoPiLOT design is a novel step forward in the development of a sensitive, user-friendly, low-resource, non-enzymatic diagnostic test.

Evaluation of Droplet Digital PCR Assay for the Diagnosis of Candidemia in Blood Samples.

Numerous studies have shown that droplet digital PCR (ddPCR) is a promising tool for the diagnosis of pathogens, especially in samples with low concentrations of pathogenic DNA. An early diagnosis of candidemia is critical for the effective treatment of patients. In this study, we evaluated the sensitivity and specificity of ddPCR assay for Candida DNA detection both in vitro by mixing fungal cells with human blood and in vivo by analyzing blood samples from infected mice and patients with suspected candidemia. The results showed that ddPCR assay could detect a minimum of 4.5 DNA copies per reaction in blood samples. ddPCR showed higher sensitivity and specificity for Candida DNA detection than traditional culture and quantitative PCR (qPCR) methods and also exhibited significantly better positive and negative predictive values than the culture and qPCR methods that were commonly used in clinical practice. Hence, our study demonstrates that ddPCR assay is a promising method for the timely diagnosis of candidemia and could be useful for monitoring the treatment of candidemia.

CRISPR-cas12a mediated SERS lateral flow assay for amplification-free detection of double-stranded DNA and single-base mutation

Lateral flow assay (LFA) is user-friendly diagnostic tools but suffering limitations in poorer sensitivities and specificities especially for double-stranded DNA and single-base mutation quantification. Here, to improve the sensitivity and specificity for the LFA based nucleic acid detection, CRISPR-Cas12a mediated Surface enhanced Raman scattering (SERS) LFA was developed. By combination of ultra-sensitive SERS tags and target-specific signal amplification ability of CRISPR-Cas12a, HIV-1 dsDNA can be directly quantified with a LOD of 0.3 fM without any pre-amplification steps, which is almost 4 orders of magnitude lower than that of traditional colorimetric LFA methods. The whole detection process can be finished less than 1 h. Moreover, based on the target specificity of Cas12a, HIV-1 single-based drug resistance mutation (M184V) can be recognized as low as 0.01%. The HIV-1 dsDNA can also be successfully detected in serum samples with good comparable of that in buffer setting. Therefore, the simple and inexpensive paper-based CRISPR-SERS strip has great potential for point-of-care testing (POCT) of nucleic acid targets especially in resource-poor or non-laboratory environments.

Detection of endothelial cell-associated human DNA reveals transplanted human bone marrow stem cell engraftment into CNS capillaries of ALS mice

Repairing the altered blood-CNS-barrier in amyotrophic lateral sclerosis (ALS) is imperative to prevent entry of detrimental blood-borne substances into the CNS. Cell transplantation with the goal of replacing damaged endothelial cells (ECs) may be a new therapeutic approach for barrier restoration. We showed positive effects of human bone marrow-derived CD34+ cells (hBM34+) and endothelial progenitor cells (hBM-EPCs) intravenous transplantation into symptomatic G93A SOD1 mutant mice on barrier reparative processes. These benefits mainly occurred by administered cells engraftment into vascular walls in ALS mice; however, additional studies are needed to confirm cell engraftment within capillaries. The aim of this investigation was to determine the presence of human DNA within microvascular ECs isolated from the CNS tissues of G93A SOD1 mutant mice treated with human bone marrow-derived stem cells. The CNS tissues were obtained from previously cell-treated and media-treated G93A mice at 17 weeks of age. Real-time PCR (RT-PCR) assay for detection of human DNA was performed in ECs isolated from mouse CNS tissue. Viability of these ECs was determined using the LIVE/DEAD viability/cytotoxicity assay. Results showed appropriate EC isolation as verified by immunoexpression of endothelial cell marker. Human DNA was detected in isolated ECs from cell-treated mice with greater concentrations in mice receiving hBM-EPCs vs. hBM34+ cells. Also, higher numbers of live ECs were determined in mice treated with hBM-EPCs vs. hBM34+ cells or media-injection. Results revealed that transplanted human cells engrafted into mouse capillary walls and efficaciously maintained endothelium function. These study results support our previous findings showing that intravenous administration of hBM-EPCs into symptomatic ALS mice was more beneficial than hBM34+ cell treatment in repair of barrier integrity, likely due to replacement of damaged ECs in mouse CNS vessels. Based on this evidence, hBM-EPCs may be advanced as a cell-specific approach for ALS therapy through restored CNS barrier integrity.

Development of a 16S metabarcoding assay for the environmental DNA (eDNA) detection of aquatic reptiles across northern Australia

A severe lack of distribution data for aquatic reptiles in northern Australia leaves many taxa vulnerable to extirpation and extinction. Environmental DNA (eDNA) technologies offer sensitive and non-invasive genetic alternatives to trapping and visual surveys and are increasingly employed for the detection of aquatic and semi-aquatic reptiles. However, these eDNA approaches have largely applied species-specific primers that do not provide a cost-effective avenue for the simultaneous detection of multiple reptilian taxa. Here, we present a mitochondrial 16S rRNA metabarcoding assay for the broad detection of aquatic and semi-aquatic reptile species. This assay is tested on water samples collected at multiple sampling sites at two tropical locations, including 12 marine and estuarine sites in Roebuck Bay, Western Australia, and four estuarine sites in Cooktown, Queensland, Australia. In total, nine reptile taxa were detected from 10 of the 16 sampled sites, including marine and freshwater turtles, aquatic, semi-aquatic and terrestrial snakes, and terrestrial skinks. However, inconsistencies in the detection of previously observed aquatic reptiles at our sampled sites, such as saltwater crocodile and sea snakes, indicated that further research is required to assess the reliability, strengths and limitations of eDNA methods for aquatic reptile detection before it can be integrated as a broad-scale bioassessment tool.

A short interspersed nuclear element-based quantitative PCR assay for simultaneous human and dog DNA detection and quantification.

This study aimed to develop a quantitative PCR assay to simultaneously quantify human and dog DNA in a multispecies mixture to inform downstream analyses in routine forensic casework and scientific research. The human target is the Alu Yb8 element, which has approximately 2000 copies per cell, and the canine target is the SINEC_Cf element, which has approximately 200,000 copies per cell. The internal positive control is a universal exogenous assay consisting of forward and reverse primers, a NED-labeled probe with an MGBNFQ quencher and a 65-bp synthetic template. Results suggest a potentially robust assay with a fast run time and a high degree of sensitivity, precision and species specificity, with direct application to domestic pet samples in veterinary genetics and forensics.

Comparative study of fluorescence and surface-enhanced Raman scattering with magnetic microparticle-based assay for target bacterial DNA detection

Magnetic microparticles (MMPs) have been extensively utilized in various assays as protein biomarkers or nucleic acid targets using a magnetic field owing to the diverse surface chemistry and ease of separation. In this study, we designed a simple MMP-based assay for target bacterial DNA with two different optical signals: fluorescence and Raman scattering. The sensitivity and multiplexing capability of both the signals were rigorously compared under optimized assay conditions. The MMP-based assay enabled the detection of multiple target bacterial DNAs with high reliability, sensitivity, and multiplexing capability, as demonstrated by the designed target DNA sequences from Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. The surface-enhanced Raman scattering (SERS)-based detection of the Escherichia coli genome DNA exhibited better sensitivity (∼30 fM) than that of the fluorescence method. Hence, the MMP-based assay in this scheme is a promising method of detecting bacteria. Further, the results of the systematic comparison between fluorescence and surface-enhanced Raman scattering will be beneficial for future designs of assay protocols with optical signals.