A Simple, Affordable, and Rapid Visual CRISPR-Based Field Test for Sex Determination of Earlier Porcine Embryos and Pork Products.

Sex selection technologies have immensely impacted swine production globally. Conventional earlier embryo sex identification methods require professional technicians and sophisticated laboratory instruments. Rapid on-site gender identification of porcine embryos and pork products remains challenging. In this study, we developed a CRISPR/Cas12a-based fluorescence visualization point-of-care sex determination test that is rapid, accurate and easy to implement on-site. The CRISPR/Cas12a assay coupled with either the polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) employs precisely designed primers and single-guide RNAs targeting the sex-determining region Y (SRY) and the zinc finger protein X-linked (ZFX) genes. PCR and LAMP amplicons were cleaved with the subsequent generation of fluorescing products detectable with portable blue and ultraviolet light transilluminators. Approximately two copies per microliter of the ZFX and SRY genes were detected using the RApid VIsual CRISPR (RAVI-CRISPR) assay. This method is a sensitive, inexpensive, versatile, and point-of-care test. The technology has other potential applications like determining the sex of diverse livestock species, detecting livestock disease-causing pathogens and evaluating the quality of meat products.

Development of a Naked Eye CRISPR-Cas12a and -Cas13a Multiplex Point-of-Care Detection of Genetically Modified Swine.

The Rapid Visual CRISPR (RAVI-CRISPR) assay employs Cas12a and Cas13a enzymes for precise gene detection in a sample. However, RAVI-CRISPR is limited in single-tube multiplex detection applications due to the lack of specific single-strand (ss) DNA-fluorescently quenched (ssDNA-FQ) and RNA-fluorescently quenched (ssRNA-FQ) reporter cleavage mechanisms. We report the development of a sensitive and specific dual-gene Cas12a and Cas13a diagnostic system. To optimize the application for field testing, we designed a portable multiplex fluorescence imaging assay that could distinguish test results with the naked eye. Herein, dual gene amplified products from multiplex recombinase polymerase amplification (RPA) were simultaneously detected in a single tube using Cas12a and Cas13a enzymes. The resulting orthogonal DNA and RNA collateral cleavage specifically distinguishes individual and mixed ssDNA-FQ and ssRNA-FQ reporters using the green-red-yellow, fluorescent signal conversion reaction system, detectable with portable blue and ultraviolet (UV) light transilluminators. As a proof-of-concept, reliable multiplex RAVI-CRISPR detection of genome-edited pigs was demonstrated, exhibiting 100% sensitivity and specificity for the analysis of CD163 knockout, lactoferrin (LF) knock-in, and wild-type pig samples. This portable naked-eye multiplex RAVI-CRISPR detection platform can provide accurate point-of-care screening of genetically modified animals and infectious diseases in resource-limited settings.

The burden and types of anaemia among HIV infected, ART-naive injection substance users in Kenya.

Introduction: Illicit substance use and HIV infection cause haematological derangements. Anaemia characterized by a reduction in the quality and quantity erythrocytes is the most common disorder in both HIV-positive persons and illicit substance users. Objective: To describe anaemia burden, types, and its association with HIV in injectable substance users in Mombasa, Kenya. Methods: This descriptive case-control study evaluated red cell indices and morphology in 494 adults. The primary outcome was anaemia. The association of anaemia with HIV in injection substance users was determined using the chi-square test. Results: The participants included 275 injection substance users (ISU), (HIV-positive, n=62 and HIV-negative, n=213); and 219 non-injection substance users (nonISU), (HIV-positive, n=33 and HIV-negative, n=186). Overall, 49% were anaemic with anaemia burden significantly differing across the groups, X2(3, N=494) =12.1, p=0.0070. Anaemia burden was higher in HIV-positive ISU compared to HIV-negative ISU (odds ratio (OR) = 1.59, 95% confidence interval (CI): 0.85, 2.96); and HIV-positive nonISU compared to HIV-negative nonISU (OR = 0.37, 95% confidence interval (CI): 0.17, 0.79). Most of the anaemia was dimorphic in both HIV-positive (ISU, 67% and nonISU, 52%) and HIV-negative (ISU, 43% and nonISU, 55%) participants. Conclusion: Infection with HIV is associated with increased risk of anaemia in injectable and non-injectable substance users. Majority of the anaemia was dimorphic suggestive of multiple aetiologies. Establishing the related aetiologies is essential for the effective treatment of anaemia. The accurate evaluation of thin blood films remains an essential tool in diagnosing an array of haematologic disorders and as a reference for further tests and patient management.

An Inexpensive CRISPR-Based Point-of-Care Test for the Identification of Meat Species and Meat Products.

The growing demand for and supply of meat and meat products has led to a proportional increase in cases of meat adulteration. Adulterated meat poses serious economic and health consequences globally. Current laboratory methods for meat species identification require specialized equipment with limited field applications. This study developed an inexpensive, point-of-care Loop-Mediated Isothermal Amplification (LAMP)-CRISPR/Cas12a colorimetric assay to detect meat species using a Texas Red-labelled single-strand (ssDNA) reporter. As low as 1.0 pg/µL of the porcine NADH4, the chicken NADH dehydrogenase subunit 2 (ND2) and the duck D-loop genes was detectable under white, blue and ultraviolet light. The test turnaround time from DNA extraction to visualization was approximately 40 min. The assay accurately detected pure and mixed-meat products in the laboratory (n = 15) and during a pilot point-of-care test (n = 8) in a food processing factory. The results are 100% reproducible using lateral flow detection strips and the real-time PCR detection instrument. This technology is fully deployable and usable in any standard room. Thus, our study demonstrates that this method is a straightforward, specific, sensitive, point-of-care test (POCT) adaptable to various outlets such as customs, quarantine units and meat import/export departments.