ABSTRACTIsothermal nucleic acid amplification tests (iNAT), such as loop-mediated isothermal amplification (LAMP), are good alternatives to polymerase chain reaction (PCR)-based amplification assays, especially for point-of-care and low resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can generate spurious amplicons, especially in the absence of target sequences, resulting in false positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets, and can accumulate mutations that will lead to inefficient primer binding and thus false negative results. Internally redundant assays targeting different regions of the target sequence can help to reduce such false negatives. Here we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on non-sequence-specific readout into assays that can be visually read using sequence-specific fluorogenic oligonucleotide strand exchange (OSD) probes. We evaluate one-pot operation of both individual and multiplex LAMP-OSD assays and demonstrate detection of SARS-CoV-2 virions in crude human saliva.

Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva. IMPORTANCE One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accurate and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes.

Spotted fever rickettsiosis plagues countries around the world. One of the deadliest of this group, Rickettsia rickettsii, responsible for Rocky Mountain spotted fever, is an emerging tickborne illness in North America. The predominant clinical diagnostic is PCR based but does not work until disease has progressed to a severe phase of infection, at which point the outcome of a full recovery is significantly decreased. An alternative, loop mediated isothermal amplification through one-step strand displacement (LAMP-OSD) assay, was developed to improve diagnostic speed and sensitivity. Synthetic dsDNA genes from the 17 kDa surface antigen precursor (AY281069.1) amplified between fifteen minutes to an hour and were detected to concentrations as low as 102 copies/μL. This RMSF LAMP-OSD assay shows promise to deliver results in just a few hours and the detection limit is potentially 100 times more sensitive than qPCR-based assays.

Hispanic women with newly diagnosed breast cancer who qualify for genetic testing based on national guidelines are often not referred to genetic counseling at the same rate as non-Hispanics patients. The reasons for the lower referral and testing rate are not currently well understood. Often patients in a minority population face diverse barriers to accessing care, particularly care that requires specialized training, is expensive, and may not be completely covered by insurance. In our study, we sought to explore the rate of referral and testing of newly diagnosed breast cancer patients in a predominantly Hispanic population at UT Health Mays Cancer Center in San Antonio where more than 60% of patients are Hispanic. Included in this study were 153 sequential new patient referrals to the breast medical oncology clinic with the diagnosis of invasive carcinoma or ductal carcinoma in situ. Current NCCN guidelines for genetic testing were utilized. Data collected included age of diagnosis, current age, gender, race, ethnicity, preferred language, stage, family history of breast, ovarian, pancreatic, or prostate cancer. It was then determined if patients met criteria, if they were referred to genetic counselors, if they attended their appointment, and if they received genetic testing. The prevalence of Hispanic women meeting criteria for genetic testing was 63.9% compared with 61.7% of non-Hispanic women. Of the Hispanic women meeting criteria for genetic testing only 71.8% received testing. Our cancer center has many processes in place that would be expected to increase the rate of genetic testing for Hispanic patients. These include having Spanish speaking staff, a robust genetic counseling team, telemedicine visits, county programs that allow unfunded patients to access care at our cancer center, and grant funding from the state of Texas to cover the additional cost of testing for unfunded patients. Despite this, about 30% of Hispanic patients that met criteria go without testing. Based on review of this data set, the two most common reasons for not completing testing was failure to recognizing that the patients meet criteria and failure to complete the extra clinic visit with the genetic counselor after referral.This study shows that Hispanic patients meet criteria for genetic testing at a slightly higher rate than Non-Hispanic patients and are at risk for not completing genetic testing even in a specialized clinic. Additional patients continue to be added to this dataset, but initial potential interventions include same time genetic counselling and testing when appropriate at the initial consultation with medical oncology or the offering of universal testing to this high-risk population. Citation Format: Allysa Lynn Garcia, Niklesh Akula, Daniel Kade Derrick, Christopher M Worrell, Simren Lakhotia, Shwetha Menon, Kate Lathrop. Optimizing genetic testing in Hispanic women with breast cancer [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 P2-09-12.