One-pot RPA-Cas12a assay for instant and visual detection of Burkholderia pseudomallei

Highlights

• An all-in-one reaction system of OPC-IVD was developed for B. pseudomallei sample-to-answer detection.

• The streamlined workflow can be completed around 30 min with proved sensitivity and specificity.

• The IAC incorporated duplex OPC-IVD allowed to check negative results.

• The instrument-free assay was successfully applied in clinical samples for POC diagnosis of melioidosis.

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis, a potentially life-threatening infectious disease, and poses public health risks in endemic areas. Due to the high mortality, intrinsic antibiotic resistance, and atypical manifestations, establishing a rapid, accurate, and sensitive identification of B. pseudomallei enables earlier diagnosis, proper treatments, and better outcomes of melioidosis. Herein, we present a One-Pot CRISPR-integrated assay for Instant and Visual Detection (termed OPC-IVD) of B. pseudomallei. The integration of recombinase polymerase amplification and CRISPR-Cas12a recognition-activated trans-cleavage, achieved a true all-in-one single-tube reaction system, initiating the amplification and cleavage simultaneously, which realized a facile sample-to-answer assay. This approach could be performed with simplified DNA extraction and completed around 30 min by holding the reaction tube in the hand. The detection limit of our OPC-IVD was determined to be 2.19 copy/uL of plasmid DNA, 12.5 CFU/mL of B. pseudomallei, and 61.5 CFU/mL of bacteria in spiked blood samples, respectively. Furthermore, the introduction of internal amplification control effectively reduced the occurrence of false negatives, which was incorporated in the reaction system, and amplified simultaneously with the target and read by naked eyes. The assay exhibited 100% accuracy when evaluated in clinical isolates and samples. The streamlined workflow of our OPC-IVD of B. pseudomallei enables a field-deployable, instrument-free, and ultra-fast approach that can be utilized by non-expert personnel in the field of molecular diagnosis of melioidosis especially in under-resourced setting.

Introduction

Melioidosis, a fatal disease caused by gram-negative bacillus B. pseudomallei, is prevalent in tropical and subtropical regions [1]. However recently, more and more sporadic cases are encountered in areas that are not endemic, as increasing international travelling and trades [2,3]. B. pseudomallei is spread to humans and animals through direct contact with the contaminated source, which contributes to a high mortality that can be up to 40% in the acute attack [4]. Especially, the septic melioidosis which can lead to death in less than 48 h if not diagnosed and treated properly [5]. In addition, no vaccine has been approved by far, and it is estimated to account for an incidence of 165,000 melioidosis cases per year (an incidence rate of 5.0 per 100,000 people at risk) with 89,000 deaths [[6], [7], [8]]. Nevertheless, the greater risk of latent infection with subsequent reactivation can occur decades after the first exposure, which was called “time bomb”, posing a serious public health threat [9]. Due to the highly variable symptoms, melioidosis is known as “great mimicker”, which causes a diagnostic dilemma in prevalent district [4]. Bacterial culture is considered as an imperfect gold standard for its time-consuming and requirement of BSL-2 enhanced laboratory, not to mention that it is susceptible to misidentify to other non-fermentative bacteria due to lack of typical biochemical characterization. Serological test appeared dissatisfactory accuracy because of the high seropositivity among healthy people in endemic regions [10], while antigen test depends on the strain serotypes and the reagent performance of antibody not to mention some patients are seronegative during acute infection, which represented certain limitations in terms of specificity and sensitivity [11,12]. Molecular diagnosis can provide the evidence of disease at the early stage of infection by detecting the specific nucleic acids of pathogens. Especially, the nucleic acid amplification-based methods have been greatly developed and increasingly considered as a gold standard for diagnosis of infectious diseases since the outbreak of COVID-19 pandemic [13]. However, most of them rely on expensive equipment, laborious process, or well-trained technicians, all of which are not suitable for easy, timely, and point of care (POC) test to help reduce the hazards of melioidosis. To date, various isothermal amplification methods, such as rolling circle amplification (RCA)-, exponential amplification reaction (EXPAR)-, nicking enzyme-assisted amplification (NEAA)-, loop-mediated isothermal amplification (LAMP)-, and recombinase polymerase amplification (RPA)-based assays have received considerable attention because of their simplicity, rapidity, and low-cost, which makes them suitable for bedside analysis [14,15]. Whereas the undesired nonspecific signal or the inhibition of amplification are still the barriers to apply them in POC molecular diagnosis.

Lately, clustered regularly interspaced short palindromic repeat (CRISPR)–based diagnostic methods collectively provide a nascent platform for the detection of pathogenic nucleic acids, such as SHERLOCK, DETECTR and HOLMES [[16], [17], [18]]. Among these, the striking feature of trans-cleavage by CRISPR-associated protein (Cas) has already been greatly explored in Cas-based detection platform, including the detection of virus, bacteria, single nucleotide polymorphism, cancer cells, exosomes, telomerase, as well as small molecules [[19], [20], [21], [22], [23], [24]]. All these achievements highlight the importance and potential applications of CRISPR-Cas as a new generation of molecular diagnosis technology with improved sensitivity and specificity [25,26]. While, these approaches depend on multiple-step manual operations constituted of template-extraction and target-amplification followed by CRISPR-mediated detection, which undoubtedly complicates the procedures, potentially increases the risk of carryover and aerosol contaminations during the amplicon transferring. Even if there have been some innovations of one-pot methods without lid opening during the detection process, most of them were physical separated, which were still cumbersome and time-consuming when need centrifugation or special-designed reaction vessel [[27], [28], [29], [30], [31]]. In addition, as diagnosis of etiologic agents involved DNA extraction from human samples which may contain amplification inhibitors or inference from the background DNA, internal amplification control (IAC) is advisable to validate the negative results. But this often demands instruments with multichannel fluorescence reader for simultaneous detection of target and IAC, which is unsuitable for POC testing with limited resource. Therefore, a new strategy that does not rely on multistep process or specialized devices is desirable for the on-site test of pathogen in molecular diagnosis.

Mitigating global infectious of melioidosis requires diagnostic tools that are sensitive, specific, and field-deployable. Here, we integrated duplex RPA with CRISPR-Cas12a to provide a true all-in-one one-pot assay, named OPC-IVD, for instant and visual detection of B. pseudomallei gDNA (Fig. 1A). In our OPC-IVD reaction, all components of RPA reaction and LbCas12a cleavage were mixed in a single-tube system from the outset, and incubated at a constant temperature, which not only avoided the operation of lid-opening for amplification products transferring, but also showed the advantage of high-efficiency and timesaving, so that the whole detection can be accomplished around 30 min including simplified DNA extraction. Furthermore, we incorporated homosapiens ACTB gene as the IAC that was abundant in human samples, into OPC-IVD system to ensure amplification validity by confirming negative results are not due to poor assay performance, but due to the absence or undetectable levels of targets. The RPA reaction with HNB dye changed color from dark blue (before) to light blue (after) if samples contained template DNA (both target and IAC), whereas no such color change would be observed in invalid samples. The target and IAC can be duplexed amplified by RPA and simultaneously observed by naked eyes with fluorophore label and colorimetric dyes, respectively. Besides, a UV torch was adopted as the illuminator, and holding the reaction tube in fist as the incubator, which were successfully applied in clinical samples for instrument-free POC diagnostics (Fig. 5C).