CRISPR RNA for detecting Burkholderia pseudomallei related genetic materials in biological samples and use method of CRISPR RNA for detecting Burkholderia pseudomallei related genetic materials in biological samples

Abstract

A CRISPR RNA (crRNA) suitable for use in a CRISPR-based ribonucleoprotein (RNP) system is provided for reacting to one or more genetic material derived from Burkholderia pseudomallei in a biological sample. The crRNA comprises a spacer region, and the spacer region is provided with one of polynucleotide sequences selected from SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7; the repeating region is arranged in front of the spacer region, and a secondary structure of the repeating region is formed; and a first extension region disposed immediately behind the spacer region.

Description

Technical Field

[0001] The present disclosure relates to an improved CRISPR RNA (crRNA) suitable for use in a CRISPR-based ribonucleoprotein (RNP) system for detecting the presence of genomic DNA of Burkholderia pseudomallei, the pathogen of melioidosis, in a subject. More specifically, the disclosed crRNA can be used in a lateral flow assay to detect the presence of Burkholderia pseudomallei in a biological sample. Background Art

[0002] Burkholderia pseudomallei is an environmental Gram-negative bacillus found primarily in soil and water and is the causative agent of melioidosis, a rapidly fatal infectious disease endemic in many tropical and subtropical countries [1-2]. If not properly diagnosed and treated, septicemic melioidosis can lead to death within 48 hours [3]. Human hosts can acquire B. pseudomallei via skin inoculation, ingestion of contaminated food or water supplies, or inhalation of aerosolized bacteria. Infected individuals can present with a variety of clinical manifestations, ranging from pneumonia to organ abscesses to sepsis [4], making clinical diagnosis challenging. Mortality rates among infected patients are high, with a reported mortality rate of 35–40% in Thailand despite antibiotic treatment [1,5]. This is in part due to delayed or missed diagnosis, which often results in delayed corrective treatment. The gold standard laboratory diagnosis of melioidosis remains culture of B. pseudomallei from clinical specimens followed by a panel of biochemical tests [6]. This diagnosis can take several days, requires an enhanced biosafety level 2 (BSL2 enhanced) laboratory, and can sometimes yield inconsistent results [4,7]. Since timely and optimal treatment of melioidosis affects the outcome of the disease [4,7,8], rapid and accurate identification of Burkholderia pseudomallei is highly desirable.

[0003] Serological diagnostics using antigens or antibodies specific for B. pseudomallei, such as indirect hemagglutination (IHA), enzyme-linked immunosorbent assay (ELISA), immunochromatographic assay (ICT), and lateral flow immunoassay (LFI), have been developed [9–16]. However, the interpretation of these serological tests has proven to be complicated in endemic areas where previous infection or previous exposure to the bacterium causes seroconversion without active infection. In addition, heterogeneity in antibody responses to B. pseudomallei has been reported in patients, necessitating the need to test for multiple antigens to confirm the results. Furthermore, some patients with culture-confirmed B. pseudomallei were seronegative during acute infection. These together limit the use of serological tests as diagnostic tools for melioidosis. In addition to serological tests, real-time polymerase chain reaction (real-time PCR)-based assays have been developed to detect B. pseudomallei DNA [17,18]. Although these methods have shown high sensitivity and specificity, real-time PCR-based assays require expensive real-time PCR machines that may not be readily available to many laboratories. As an alternative, isothermal DNA amplification, such as loop-mediated isothermal amplification (LAMP)

[19] and recombinase polymerase amplification (RPA) [20, 21], have been developed to overcome this reliance on expensive equipment. Both assays have demonstrated very sensitive detection. For example, the limit of detection (LOD) of RPA is between 25 and 50 copies of B. pseudomallei genomic DNA. Although promising, these assays are based on primers designed to target genomic loci, such as the type 3 secretion system-1 (T3SS-1) of a few selected reference strains. Their coverage of detection across the broader population of B. pseudomallei is currently unknown. In addition, the specificity of the RPA assay remains to be further determined, as it can tolerate some mismatches in the primer sequence.

[0004] Clustered regularly interspaced short palindromic repeats (CRISPR) are an adaptive defense system in bacteria and archaea that provide sequence-specific immunity against invading nucleic acids, such as bacteriophages or plasmids

[22] . CRISPR loci contain remnants of genetic material from past infections and use them for RNA-guided endonucleases to combat future infections. This RNA-guided endonuclease is called a CRISPR-Cas complex and consists of CRISPR RNA (crRNA) and a CRISPR-associated (Cas) nuclease. The complex recognizes the target site through crRNA-mediated base pairing complementarity before initiating cleavage. Various CRISPR-Cas systems have been identified, each with unique properties for nucleic acids [23,24]. One prominent system is CRISPR-Cas9, which has been used for precise genome editing in research and gene therapy

[25] and as a potential treatment for infectious diseases such as human immunodeficiency virus (HIV) and hepatitis B virus (HBV) [26,27]. CRISPR-Cas12a and -Cas13a are among the most recently discovered members of the CRISPR-Cas family that recognize DNA and RNA targets, respectively. Their initial target recognition activates nonspecific endonucleases that indirectly cleave other non-target nucleic acids in their vicinity [28,29]. This side-branch cleavage property of both CRISPR-Cas systems has been used as a biosensor to induce cleavage of fluorescent nucleic acid probes, enabling sensitive and quantitative readout of the actual target nucleic acid

[30] . By combining this property of CRISPR-Cas12a or -13a with another signal enhancement step such as metal-enhanced fluorescence (MEF), autocatalytically driven feedback amplification, or isothermal preamplification of nucleic acids, CRISPR-Cas systems can be used as detection platforms with excellent sensitivity and specificity [31-34]. For example, detection assays called DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) couple isothermal preamplification of nucleic acids with CRISPR-Cas12a and -Cas13a systems, respectively. In view of this, a simplified and time-saving melioidosis diagnostic tool or kit can be developed using CRISPR-Cas-based technology to address some of the shortcomings found in current clinical practice mentioned above. Summary of the invention

[0005] The present disclosure aims to provide one or more engineered or modified crRNAs that can be used to detect the presence of Burkholderia pseudomallei-related or specific genetic material in a biological sample. Based on the presence or absence of Burkholderia pseudomallei-related genetic material in the biological sample, the present disclosure allows at least a preliminary diagnosis of melioidosis in a subject from which the biological sample was derived to be obtained.

[0006] A further object of the present disclosure is to provide one or more crRNAs operable in a CRISPR-based RNP system for detecting genetic material associated with Burkholderia pseudomallei in a biological sample. The CRISPR-based RNP system facilitates the detection of genetic material associated with Burkholderia pseudomallei with a relatively short processing time compared to other common clinical or laboratory methods.

[0007] Nevertheless, another object of the present disclosure is to provide a method for running a biological sample utilizing the above-described crRNA and assembled CRISPR-based RNP system to identify or detect the presence of Burkholderia pseudomallei-related genetic material therein and subsequently infer at least a preliminary diagnosis regarding a potential infection associated with melioidosis.

[0008] The present disclosure satisfies at least one of the above objectives in whole or in part, wherein one of the embodiments of the present disclosure is a single guide RNA (crRNA) suitable for use in a CRISPR-based ribonucleoprotein (RNP) system for responding to one or more genetic materials derived from Burkholderia pseudomallei in a biological sample. The crRNA comprises a spacer region having one of the polynucleotide sequences selected from SEQ ID 3, SEQ ID 5, and SEQ ID 7; a repeat region before the spacer region, forming its secondary structure; and a first extension region disposed immediately after the spacer region.

[0009] For further embodiments, the spacer is configured to hybridize to a complementary sequence residing in the genetic material, and the complementary sequence is preceded by a pre-spacer adjacent motif (PAM) comprising a TTTV sequence with V being a non-T nucleotide.

[0010] For more embodiments, the crRNA includes a second extension region having a Gs sequence, and GGG is preferably arranged in front of the repeat region.

[0011] For more embodiments, the repeat region comprises a polynucleotide sequence of SEQ ID No. 9 or SEQ ID No. 10, or a polynucleotide sequence having at least 80% sequence identity with SEQ ID No. 9.

[0012] For more embodiments, the first extension region comprises a polynucleotide sequence of AAAGGAA.

[0013] Preferably, in some embodiments, the CRISPR-based RNP system is a CRISPR-Cas12a RNP system, a CRISPR-Cas12b RNP system or a CRISPR-Cas13 RNP system.

[0014] Another aspect of the present disclosure relates to a method for detecting the presence of one or more genetic materials of Burkholderia pseudomallei obtained from a biological sample, the method comprising the following steps: providing one or more genetic materials of Burkholderia pseudomallei; assembling a CRISPR-based RNP system, the assembled CRISPR-based RNP system comprising a crRNA, whose spacer has one of the polynucleotide sequences selected from SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7; reacting the genetic material with the assembled CRISPR-based RNP system along with multiple reporter probes in a buffer solution, the CRISPR-based RNP system being configured to activate the cleavage of the reporter probe so as to emit a fluorescent signal when hybridized to a polynucleotide sequence complementary to the spacer located within the one or more genetic materials; and detecting the emitted signal to confirm the presence of Burkholderia pseudomallei in the provided biological sample.

[0015] In some embodiments of the disclosed methods, the detecting step further comprises subjecting a portion of the reacted buffer solution to a lateral flow assay to capture the emitted signal. The reaction buffer may contain the end product of a reaction between a preassembled CIRSPR RNP system and genetic material (e.g., a partial or complete genome of Burkholderia pseudomallei DNA). Preferably, the lateral flow assay may be a dipstick for capturing and then visualizing the detectable portion or signal released from the signaling probe.

[0016] In some embodiments of the disclosed methods, the crRNA further comprises a repeating region before the spacer region that forms its secondary structure, and the repeating region comprises a polynucleotide sequence of SEQ ID No. 9 or SEQ ID No. 10.

[0017] In some embodiments of the disclosed method, the crRNA also includes a first extension region arranged immediately after the spacer region, and the first extension region includes a polynucleotide sequence of AAAGGAA. When the crRNA is hybridized to a target sequence located in the genetic material of the biological sample, the addition of the first extension is conducive to the side branch cutting activity. In addition, in some embodiments of the disclosed method, the crRNA also includes a second extension region having a GGG sequence arranged before the repeat region.

[0018] Preferably, in several embodiments of the disclosed methods, the CRISPR-based RNP system is a CRISPR-Cas12a RNP system derived from the Lachnospiraceae bacterium ND2006.

[0019] In many embodiments of the disclosed system, the spacer is configured to hybridize to a complementary sequence residing in the genetic material, and the complementary sequence is preceded by a pre-spacer adjacent motif (PAM) comprising a TTTV sequence, wherein V is a non-T nucleotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1A A schematic diagram showing the CRISPR-Cas12a developed in the present disclosure recognizes its target DNA;

[0021] Figure 1B Phylogenetic diagram of the global population of Burkholderia pseudomallei collected between 1935 and 2018 (n=3341) is shown, with the outer ring representing the major lineages, represented as Burkholderia Bayesian clusters (bb) 1 to 22, and the dots located in the inner ring highlighting the 30 representative genomes used for CRISPR-Cas12a target scanning;

[0022] Figure 1C is a schematic diagram of the bioinformatics analysis used to identify Burkholderia pseudomallei-specific CRISPR-Cas12a target sites, including CRISPR selection and CRISPR filtering;

[0023] Figure 2A A schematic diagram of the DETECTR assay is shown;

[0024] Figure 2B is a graph showing the collateral cleavage activity of crBP34;

[0025] Figure 2C is a graph showing the collateral cleavage activity of crBP36;

[0026] Figure 2D is a graph showing the collateral cleavage activity of crBP38;

[0027] Figure 3A is a heat map showing the fluorescence signal generated by the crBP34-DETECTR assay;

[0028] Figure 3B is a heat map showing the fluorescent signal generated by the real-time PCR assay;

[0029] Figure 3C is a schematic diagram of a lateral flow assay developed by the present disclosure, wherein RPA and CRISPR reactions are performed similarly to Figure 2A , except that the FAM-quencher probe was replaced by the FAM-biotin probe (FB);

[0030] Figure 3Dis a graph showing that lateral flow dipstick readings retain the sensitivity of the crBP34-DETECTR assay; and

[0031] Figure 4 is a graph showing the results of crBP34-DETECTR assays against various clinical isolates including Burkholderia pseudomallei. DETAILED DESCRIPTION

[0032] Hereinafter, the present disclosure shall be described in terms of preferred embodiments and by reference to the accompanying description and drawings. However, it should be understood that the description with reference to the preferred embodiments and drawings of the present disclosure is only for the purpose of facilitating the discussion of the various disclosed embodiments, and it is contemplated that various modifications may be devised by those skilled in the art without departing from the scope of the appended claims.

[0033] The term "polynucleotide" or "nucleic acid" as used herein refers to, for example, mRNA, RNA, cRNA, cDNA or DNA. The term generally refers to an oligonucleotide greater than 30 nucleotide residues in length.

[0034] The term "genetic material" as used in this specification refers to all or part of the genome of Burkholderia pseudomallei that has been extracted, replicated and / or amplified using known methods from an organism, preferably obtained from a body fluid of a subject or patient. The organism obtained from the body fluid may be further cultured to increase the number of Burkholderia pseudomallei and the replication of the genetic material associated therewith. In addition, the genetic material may be subjected to one or more pre-treatment steps prior to performing the disclosed methods.

[0035] The term "CRISPR RNA" or "crRNA" used herein refers to an RNA molecule that plays a key role in the CRISPR-Cas system, acting as a guide molecule to guide the Cas protein to a specific DNA sequence for precise targeting. It typically includes a repeat sequence and a spacer region, which promotes interaction with the homologous Cas enzyme and the target DNA, respectively. The spacer region is typically 17-24 nucleotides in length, complementary to the target DNA, and is preferably a conserved nucleotide sequence retained in the genetic material of Burkholderia pseudomallei.

[0036] Unless otherwise indicated, the terms "crBP32", "crBP33", "crBP34", "crBP35", "crBP36", "crBP37", "crBP38" and "crBP39" used in the detailed description, especially in the examples section, shall refer to SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8, respectively.

[0037] According to one aspect of the present disclosure, a crRNA suitable for use in a CRISPR-based RNP system is disclosed for reacting to one or more genetic materials derived from Burkholderia pseudomallei in a biological sample. The crRNA basically comprises a spacer region having one of the polynucleotide sequences selected from SEQ ID No.3, SEQ ID No.5, and SEQ ID No.7; a repeat region is provided before the spacer region to form its secondary structure; and a first extension region is provided immediately after the spacer region. The Burkholderia pseudomallei in the biological sample can be pre-treated or treated in advance, such as cultured in a suitable culture medium to increase the number of Burkholderia pseudomallei and the genetic material associated therewith. For some embodiments, the bacteria of Burkholderia pseudomallei cultured and isolated from a patient or subject are biological samples. For more embodiments, the biological sample can be a body fluid, such as blood collected from a subject or patient, containing a sufficient number or amount of genetic material of Burkholderia pseudomallei for reacting with an established CRISPR-based RNP system using the disclosed crRNA. Likewise, the body fluid can be pre-treated to concentrate the genetic material of Burkholderia pseudomallei and / or remove any components that may adversely affect CRISPR traction. In some embodiments, the genes and / or non-coding sequence fragments of the genetic material of Burkholderia pseudomallei that are complementary to the spacer sequence can also be first amplified by a thermal cycling process to obtain a high concentration of amplicons carrying the complementary sequence, and then the amplicons are reacted with the CRISPR-based RNP system.

[0038] It should be noted that each of SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 in the crRNA (corresponding to crBP34, crBP36 and crBP38 in the partial description, respectively) is capable of hybridizing to a complementary sequence located in a conserved region of DNA in more than 90% of known Burkholderia pseudomallei strains worldwide, as shown in the examples given below. Utilizing these sequences that are highly specific to Burkholderia pseudomallei, the constructed crRNA and the CRISPR-based RNP system assembled to the crRNA become effective tools for searching and identifying the presence of Burkholderia pseudomallei in biological samples, thereby inferring at least a preliminary diagnosis of Burkholderia pseudomallei infection.

[0039] According to several preferred embodiments, the CRISPR-based RNP system is a CRISPR-Cas12a RNP system or a CRISPR-Cas12a RNP complex. Cas12a ribonuclease is an RNA-guided nuclease that, once activated, has a side branch cutting ability and is located in an environment that provides single-stranded deoxyribonucleic acid ssDNA. Figure 1A As shown, the Cas12a protein (grey leaves) needs to interact with crRNA, which consists of two basic elements - a repeat region and a spacer region. The repeat region is the structural scaffold of crRNA and is required for crRNA-Cas12a interaction, while the spacer region is used for target recognition.

[0040] Additionally, in some embodiments, the first extension comprises a polynucleotide sequence of AAAGGAA. The first extension added in these embodiments has shown improved collateral cleavage activity, thereby enhancing the detectability of the signal or signal portions released when identifying the presence of Burkholderia pseudomallei genetic material in a biological sample. As described above, the first extension is located immediately after the spacer or at the 3' end of the spacer.

[0041] Another effort of the present disclosure to improve the specificity of the disclosed crRNA to Burkholderia pseudomallei is to identify a specific pre-spacer adjacent motif (PAM) associated with a sequence complementary to the spacer located in the genetic material of Burkholderia pseudomallei. More specifically, the spacer is configured to hybridize on a complementary sequence in the genetic material, and the complementary sequence is preceded by a PAM with a TTTV sequence, wherein V is a non-T nucleotide. The disclosed crRNA utilizes the PAM sequence as an additional measure to verify that the complementary sequence present in the genetic material is indeed derived from Burkholderia pseudomallei, rather than from other common causes of bacterial infection, such as Streptococcus pyogenes, Streptococcus epidermidis, Klebsiella pneumoniae, Streptococcus agalactiae, Pseudomonas aeruginosa, Bacillus thailandensis, Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii. PAM is a prerequisite for activating the CRSPR-based RNP system to initiate trans-cutting of the complementary sequence.

[0042] In order to achieve the attachment of crRNA to the Cas endonuclease, the repeat region forming the scaffold structure preferably comprises a polynucleotide sequence of SEQ ID No. 9. The polynucleotide sequence of SEQ ID No. 9 is naturally folded into a secondary structure to adaptively anchor the entire gRNA or crRNA to the Cas endonuclease, thereby assembling or establishing a CRISPR-based RNP system. In more embodiments, the crRNA includes a second extension region having a GGG sequence arranged before the repeat region.

[0043] As described above, another aspect of the present disclosure relates to a method for detecting the presence of one or more genetic materials of Burkholderia pseudomallei obtained from a biological sample. Any DNA extraction procedure can be performed to obtain DNA extracts that represent the genetic material of each pathogen. Preferably, the disclosed method includes the steps of providing one or more genetic materials of Burkholderia pseudomallei; assembling a CRISPR-based RNP system, the assembled CRISPR-based RNP system including crRNA, whose spacer has one of the polynucleotide sequences selected from SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7; reacting the genetic material with the assembled CRISPR-based RNP system along with multiple reporter probes in a buffer solution, the CRISPR-based RNP system is configured to activate the reporter probe so as to emit a signal when hybridized to a polynucleotide sequence complementary to the spacer located in the one or more genetic materials; detecting the emitted signal to confirm the presence of Burkholderia pseudomallei in the biological sample.

[0044] Similarly, prior to reacting with the assembled CRISPR-based RNP system, the Burkholderia pseudomallei in the biological sample of the disclosed method can be pre-treated or pre-treated, such as cultured in a suitable culture medium, to increase the number of available Burkholderia pseudomallei, thereby increasing the amount of genetic material associated therewith. The Burkholderia pseudomallei bacteria cultured and isolated from a patient or subject can be the biological sample in some embodiments of the disclosed method. For more embodiments, the biological sample can be a body fluid, such as blood collected from a subject or patient, containing a sufficient number or amount of Burkholderia pseudomallei genetic material for reacting with an established CRISPR-based RNP system using a disclosed crRNA.

[0045] The method of the present disclosure may also include the steps of amplifying nucleotide sequence fragments complementary to SEQ ID No.3, SEQ ID No.5, and SEQ ID No.7 from the genetic material to produce a plurality of amplicons; and reacting the amplicons with the assembled CRISPR-based RNP system along a plurality of signal probes in a buffer solution, rather than directly using the genetic material in certain embodiments. Although these embodiments may incur additional time or cost compared to directly using the genetic material of Burkholderia pseudomallei contained in the biological sample, the additional steps may become convenient when the amount of genetic material of Burkholderia pseudomallei is relatively low, especially when the biological sample obtained is limited. In particular, in an isothermal amplification reaction (such as recombinase polymerase amplification (RPA), loop-mediated isothermal amplification reaction (LAMP), or other thermal cycling processes known in the art), a fragment in the genetic material containing the CRISPR-based RNP system target site in the pathogen genomic DNA is pre-amplified by flanking primers.

[0046] According to many preferred embodiments of the disclosed method, the crRNA further comprises a repeat region before the spacer region, forming its secondary structure. More preferably, the repeat region comprises a polynucleotide sequence of SEQ ID No.9 or SEQ ID No.10. In addition, in some embodiments, the crRNA comprises a second extension region having a GGG sequence arranged before the repeat region.

[0047] According to more embodiments of the disclosed method, the crRNA further comprises a first extension region arranged immediately after the spacer region. The first extension region should comprise a polynucleotide sequence of AAAGGAA. As previously described, adding the first extension to the crRNA will result in better collateral cleavage activity toward the signal probe, thereby enhancing the detectability of the signal released when identifying the presence of Burkholderia pseudomallei genetic material in a biological sample.

[0048] In some embodiments of the disclosed methods, the CRISPR-based RNP system is a CRISPR-Cas12a RNP system or a CRISPR-Cas12a RNP complex. Cas12a ribonuclease is an RNA-guided nuclease that, once activated, has side branch cutting capabilities and is used as part of a signal probe or reporter probe in a disclosed method in an environment that provides single-stranded nucleic acid ssDNA.

[0049] It should be noted that the multiple signal probes or reporter probes of the disclosed method are ssDNA or single-stranded polynucleotides, and the detectable portion and the corresponding quencher are respectively connected to the 5' end and 3' end of the single-stranded probe. The detectable portion can be 5'6-fluorescein (FAM), Cy 3, HEX (hexachlorofluorescein) or ROX (6-carboxyl-X-rhodamine), although FAM is used in those more preferred embodiments. The quencher is preferably a non-fluorescent molecule, such as IABkFQ, DABCYL or BHQ, which is shaped to suppress the signal released from the detectable portion, unless after the crRNA complementary sequence in the recognition genetic material is removed from the ssDNA or ssRNA by the side branch cutting of the activated CRISPR-based RNP system. In particular, adding a sufficient amount of Burkholderia pseudomallei genetic material to the assembled CRISPR-Cas12a RNP complex and FAM-quencher ssDNA probe (FQ) in the CRISPR reaction will cause the assembled CRISPR-RNP complex to recognize the target sequence, and the CRISPR-RNP complex self-activates its trans-cleavage (side branch cleavage) activity to cut the signal probe. This further causes the FAM fluorophore to be cut from its quencher, thereby generating a fluorescent signal that can be detected by a fluorometer or the like, thereby allowing the method of the present disclosure to confirm the presence of Burkholderia pseudomallei genetic material in a biological sample.

[0050] In addition, further embodiments of the disclosed methods preferably eliminate the requirement for expensive laboratory setups (such as fluorometers). Preferably, the method of the present disclosure also includes the step of subjecting a portion of the reaction buffer to a lateral flow assay to capture the emission signal. In order to achieve signal visualization by lateral flow assay, subtle changes must be made to the signal probe or reporter probe. In some embodiments, the quencher is replaced by biotin to achieve signal visualization. The signal probe or reporter probe for lateral flow assay is preferably FAM-biotin ssDNA. In more detail, the lateral flow assay is a lateral flow test ruler, which generally includes an absorption platform carrying a sample pad portion, a positive test portion or a positive test line, and a control portion (or an internal control line). Preferably, the sample pad portion is impregnated with a gold-conjugated anti-FAM antibody. The control portion is a portion impregnated with streptavidin, while the positive test portion is embedded with anti-IgG. It is worth noting that once the positive and / or control portion accumulates a sufficient amount of gold conjugate, the gold conjugate that can be chemically bound to FAM by the anti-FAM antibody is a reagent that imparts a visual signal observed on the positive and / or control portion.

[0051] In practice, the disclosed method directly immerses the sample pad of the lateral flow test strip into a buffer solution containing the end product of the CRISPR reaction. The sample pad contains a gold-conjugated anti-FAM antibody. In the absence of Burkholderia pseudomallei genetic material, the signal probe remains intact, and the gold conjugate bound to FAM is trapped in the control portion only due to the reaction between the impregnated streptavidin and the biotin on the intact signal probe. Therefore, only one band can be seen in the control portion. On the other hand, in the presence of Burkholderia pseudomallei genetic material, the side branch activity of CRISPR-Cas12a degrades the probe, releasing the FAM-gold-conjugated antibody complex from biotin. The released FAM-gold-conjugated antibody can diffuse through the control portion without being trapped in streptavidin until it reaches the positive test portion and binds to the anti-IgG coating, thereby generating a visual clue indicating the presence of Burkholderia pseudomallei genetic material in the biological sample. More specifically, if the biological sample contains genetic material from Burkholderia pseudomallei, two lines will be visible, one in the positive test portion and one in the control portion (because not all reporter probes will be cleaved). Lateral flow assays are incorporated into the methods of the present disclosure to allow the user to read the results of the CRISPR reaction without the need for additional equipment, such as a fluorometer or the like.

[0052] As previously described, the disclosed method further verifies that the genetic material used is indeed from Burkholderia pseudomallei, and not another pathogen with a similar genetic makeup. Thus, the spacer is configured to hybridize with a complementary sequence in the genetic material, and the complementary sequence is preceded by a pre-spacer adjacent motif (PAM) comprising a TTTV sequence, wherein V is a non-T nucleotide.

[0053] Notably, the inventors of the present disclosure have developed a method that is highly sensitive and specific for detecting Burkholderia pseudomallei genomic DNA in biological samples using a CRIPSR RNP system (preferably CRISPR-Cas12a). The currently developed detection platform is highly specific for Burkholderia pseudomallei as it detects all clinical isolates of endemic Burkholderia pseudomallei while distinguishing between human and other pathogens, including its closely related species Burkholderia thailandensis, as demonstrated by the examples given below. The adjustment of the lateral flow test strip or assay for reading in the disclosed method allows the disclosed method, as a detection platform, to be performed in a routine laboratory without the need for specific instrumentation. The disclosed method, particularly when used in conjunction with a lateral flow assay, will facilitate immediate or field-deployed diagnosis of Burkholderia pseudomallei.

[0054] Example 1

[0055] pMBP-LbCas12a (from Lachnospiraceae bacterial species) was a gift from Jennifer Doudna (Addgene, USA, #113431). The plasmid was transformed into Escherichia coli Rosetta 2 (DE3) (Novagen, USA, #70954). 500 mL of Luria-Bertani (LB) culture was cultured at 37 ° C until OD600 reached 0.4-0.6. MBP-LbCas12a expression was induced by culturing the culture overnight at 16-18 ° C at a concentration of 0.1 mM IPTG. Bacterial cells were obtained by centrifugation, washed with PBS and resuspended in 40 mL of lysis buffer (1000 mM NaCl, 20 mM imidazole, 20 mM Tris-HCl, 5% glycerol, 1 mM DTT and 1 mM PMSF). The cells were sonicated for 3 minutes at a frequency of 20 kHz and an amplitude of 60% using a Vibra-Cell VCX 500 sonicator (Sonics & Materials, USA) in an ice bath, using a 1 second pulse and a 7 second rest. The cell lysate was clarified by centrifugation at 20,000 g for 1 hour at 4°C and filtered through a 0.22 um PES membrane. All protein purification steps were performed using an AKTA Pure 25M1 FPLC system (GE Healthcare, Sweden). First, the purified protein was bound to a 5 mL HisTrap HP column (GE Healthcare, Sweden, #17524802) with His binding buffer (20 mM Tris-HCl pH 7.5, 1000 mM NaCl, 5% glycerol, 1 mM DTT, 20 mM imidazole), washed with a wash buffer (20 mM Tris-HCl pH 7.5, 1000 mM NaCl, 5% glycerol, 1 mM DTT, 50 mM imidazole), and eluted with an elution buffer (20 mM Tris-HCl pH 7.5, 1000 mM NaCl, 5% glycerol, 1 mM DTT, 300 mM imidazole). The peak fraction was collected and dialyzed overnight in a dialysis buffer (20 mM Tris-HCl pH 7.5, 125 mM NaCl, 5% glycerol, 1 mM DTT, 1 mM PMSF). Soluble protein was recovered and loaded onto a 5 mL HiTrap SP HP cation exchange column (GE Healthcare, Sweden, #17115201), pre-equilibrated with binding buffer (20 mM Tris-HCl pH 7.5, 125 mM NaCl, 5% glycerol).The column was washed with binding buffer and the protein was eluted with ion exchange buffer (20mM Tris-HCl pH 7.5, 5% glycerol, 125–2000mM NaCl gradient). The fractions containing MBP-LbCas12a were combined, concentrated and injected into a HiLoad 16 / 600 Superdex 200pg size exclusion column (GE Healthcare, Sweden, #28989335), pre-equilibrated with gel filtration buffer (50mM Tris-HCl pH 7.5, 500mM NaCl, 5% glycerol). The protein was eluted with the same buffer. The fractions containing MBP-LbCas12a were concentrated to approximately 500μl. Glycerol and DTT were added to reach final concentrations of 20% v / v and 2mM, respectively. The protein was aliquoted, snap frozen in liquid nitrogen and stored at -80°C.

[0056] Example 2

[0057] All command codes used are open source codes on GitHub (https: / / github.com / henrikroslund / crispr-cas12a / releases / tag / BP_V2). In the first step, using CRISPR selection, one of the 30 representative Burkholderia pseudomallei genomes, namely bb2, was used to generate an initial pool of all possible LbCas12a target sites by scanning TTTVN1...N 20 (PAM is in bold; V is a non-T nucleotide and N1 to N20 are spacer sequences of any nucleotide). Duplicate target sites or those with spacers containing tetranucleotides or with a GC content exceeding 40–65% were removed. This generated 26,661 candidate target sites that were subsequently aligned to 29 other representative Burkholderia pseudomallei genomes. 15,448 candidate target sites were found to be 100% conserved across all 30 genomes and were processed through a CRISPR filtering step to eliminate potential cross-reactivity with other pathogens. First, a pool of CRISPR-Cas12a target sites from 1,071 complete genomes of non-B. pseudomallei was generated TTTNN1...N 20 The PAM position 4 was relaxed, thus creating a weaker cross-reactive target site. The candidate target site from Burkholderia pseudomallei was compared with the candidate target site from the strain using only N1 to N 20The target sites of non-B. pseudomallei pathogens were aligned, and candidates with 100% match were removed. This initial filtering step significantly reduced the number of candidate target sites for B. pseudomallei to 1982. The resulting candidates were further filtered out by remapping from non-B. pseudomallei pathogens to the target site pool described above. Mappability was defined as the number of candidate target sites between N1 and N2. 20 Mapped candidates were analyzed for mismatches relative to their cross-reactive target sites. Candidates that failed to meet the following “mismatch criteria” at any mapped site were immediately removed: (i) 2 consecutive ones in the seed (N1 to N6), or (ii) 3 or more in the seed, or (iii) N7 to N8. 20 The remaining candidates and those that could not be mapped to any cross-reactive target sites were considered as final candidates (Table 1). The spacer region (excluding PAM) was later engineered into crRNA.

[0058] Table 1

[0059]

[0060]

[0061] Example 3

[0062] The DNA template was synthesized and desalted by IDT DNA Technology (Singapore). All crRNAs were in vitro transcribed from annealed oligonucleotide templates using the HiScribeT7 High Yield RNA Synthesis Kit (NEB, USA, #E2040S) according to the manufacturer's protocol. DNAse I (RNase-free) (NEB, USA, #M0303S) was added to the reaction, and the tube was incubated at 37°C for 20 minutes to degrade the DNA template. The crRNA was purified in 12% v / v urea-PAGE (29:1) (in 7.5M urea, 0.5x TBE), eluted in gel elution buffer (300mM sodium acetate pH 5.2, 1mM EDTA pH 8.0, 0.1% v / v SDS), and precipitated in ethanol. The crRNA was resuspended in folding buffer (10mM Tris-HCl, pH 7.5, 50mM NaCl), heated in a 90°C metal heating block for 2 minutes, and slowly cooled at room temperature. The crRNA was aliquoted and stored at -80 °C until use.

[0063] Example 4

[0064] A global collection of Burkholderia pseudomallei (n=3341) was assembled from public databases and other short-read sequence data. When assemblies were not available, short sequences were assembled de novo using Velvet, resulting in a median of 136 contigs (range 60-434) and an average total length of 7140818 bps (minimum = 6989389 bps, maximum = 7441976 bps). Gene prediction and annotation of the assembly were performed using Prokka. An average of 5850 (range 5685-6275 per genome) predicted coding sequences (CDS) were assigned, and the assembly fell within a similar range of 6332 CDS and 7247547 bps length reported in the reference genome K96243. Population structure was defined as previously described using three independent methods—PopPUNK, phylogenetic trees, and multidimensional scaling. All three methods showed high agreement and resolved the Burkholderia pseudomallei population into 324 lineages, many of which were singletons or lineages with a small number of isolates. The disclosure indicated that there were 22 major lineages that dominated the global Burkholderia pseudomallei population (2429 / 3341 isolates, 73% of the dataset).

[0065] To ensure that CRISPR-Cas targets encompassed the genetic diversity of the entire population, candidate genomes were selected to represent each major lineage (Burkholder Bayesian clusters (bb) 1 to 22 ( Figure 1B ), except that bb3 and bb4 share a close ancestor with bb5, of which one representative was selected to represent the three lineages. The inventors of the present disclosure also randomly selected candidate genomes outside the main lineages. A total of 30 genomes were selected. On average, each candidate could be assembled into 92 contigs, providing a fairly long conserved region for searching CRISPR-Cas targets. PopPUNK phylogenetic tree ( Figure 1B ) visualized using the Tree of Life tool and annotated with metadata.

[0066] Example 5

[0067] In silico validation of CRISPR-Cas targets was performed by mapping the CRISPR-Cas12a designed RPA primers and target sequences against an assembly of 3341 Burkholderia pseudomallei genomes (S1 Table) using BLAT v.36. To enable mapping of low-complexity sequences, the command “blat -minScore=10 -tileSize=8 <assembly><candidate target><out.psl>Since CRISPR-Cas12a recognition appears to be flexible at position 4 of the PAM and appears to allow for the 20 The current disclosure further allows BLAT results to tolerate two types of mismatches. For the sequence target TTTVN1N2N3...N 20 , the analysis allows for (i) mismatches at position V; or (ii) mismatches at position N1 to N 20 mismatch at any position of ; or a combination of (i) and (ii). To test the possible cross-reactivity of these CRISPR-Cas12a target sequences with human host DNA or other bacterial species, the inventors also mapped them against the human reference genome GRCh38

[35] and 40,827 bacterial reference genomes archived in the NCBI repository

[36] (access date May 5, 2022). To reduce the computational load, searches were performed using NCBI web BLASTN, and the results were filtered to allow (i) mismatches at position V; or (ii) mismatches from N1 to N 20 None of the hits exceeded the filtering threshold and are unlikely to be recognized by CRISPR Cas12a because N1 to N 20 A single mismatch already significantly reduces the function of the enzyme.

[0068] CRISPR-Cas12a is an RNA-guided ribonucleoprotein (RNP) complex that recognizes its target DNA through base pairing complementarity [37, 38]. Target recognition and cleavage convert the RNP complex into a nonspecific nuclease that cleaves single-stranded DNA (ssDNA) in the extracellular side branches

[29] . This side branch cleavage property and programmability of CRISPR-Cas12a provide adaptability for highly specific and sensitive DNA detection [29, 31]. Therefore, the present disclosure aims to apply this CRISPR diagnostic platform to the detection of Burkholderia pseudomallei DNA.

[0069] The key to the specificity of CRISPR-Cas12a-based detection is the crRNA spacer, which forms base pairs with the target DNA ( Figure 1A ). Since Burkholderia pseudomallei is highly recombinant and its genomic content is constantly recombining [39-41], a well-designed spacer is required to target DNA sequences present in all Burkholderia pseudomallei to ensure high detection coverage. Therefore, the present disclosure conducted a phylogenetic analysis of 3341 Burkholderia pseudomallei genomes from global collections. This analysis divided the Burkholderia pseudomallei population into 22 major lineages ( Figure 1B We then selected one representative from each major lineage and eight additional external representatives for our in-house bioinformatics pipeline, which searches for CRISPR-Cas12a candidate target sites ( Figure 1C ).

[0070] In this pipeline, the inventors of the present disclosure first generated a pool of optimal target sites that were common in 30 representative genomes of Burkholderia pseudomallei. These target sites contained TTTV (V is A, C, or G) as the 5'-end of the protospacer adjacent motif (PAM), followed by a 20-nt long spacer ( Figure 1A ). The resulting candidate target sites were then filtered through 1071 genomes of various bacterial pathogens that cause frequent infections or are closely related to Burkholderia pseudomallei, including Burkholderia cepacia, Burkholderia mallei, and Burkholderia thailandica. The candidate sites generated in the last step are either unique to Burkholderia pseudomallei or contain significant mismatches with those cross-reactive pathogens; both are likely to be highly specific target sites for CRISPR-Cas12a. The results showed that after executing the bioinformatics pipeline, the inventors retrieved 8 elite target sites, each of which was found as a single copy in the Burkholderia pseudomallei genome (Table 1). These target sites are located in the coding and non-coding regions of the genome. By computer searching for potential cross-reactive sites against the NCBI genome database

[42] (data acquired on May 5, 2022), the present disclosure further confirmed the high specificity of all target sites, with no cross-reactivity with humans

[35] or other bacterial genomes

[36] . In contrast, when analyzing the coverage of these eight target sites in the global population of B. pseudomallei, they were all found in at least 97% of the populations (Table 1).

[0071] Example 6

[0072] RPA reagents were purchased from TwistDx (USA). As recommended by TwistDx, all RPA primers were 35 nt in length and the amplicon length was less than 500 base pairs (ideally 100–200 base pairs). The secondary structures and homo / heteroduplexes of these primers were manually designed and checked using RNAfold (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi) and IDTOligoAnalyzer (https: / / www.idtdna.com / pages / tools / oligoanalyzer), respectively. Primers were synthesized and purified by standard desalting by Macrogen (Korea). TwistAmp Basic (TwistDx, USA, #TABAS03KIT) and TwistAmp Liquid Basic (TwistDx, USA, #TALQBAS01) were used in this study according to the manufacturer’s protocol with the following modifications: (i) the total volume of the RPA reaction was adjusted to 30–50 μl and (ii) incubation was performed at 39 °C for 30 min. Figure 2B , 2C and 2D), approximately 10,000 replicates of bacterial genomic DNA were used in each reaction. The replicate numbers were approximated based on the median genome size of each bacterial species. For sensitivity testing ( Figure 3A and 3B ), the exact copy number of the genomic DNA stock of Burkholderia pseudomallei (isolate 13D) was determined by real-time PCR with purified standards. The genomic DNA was then diluted to an appropriate concentration and immediately used in the RPA reaction. Figure 4 ), 1 ng DNA was used in each RPA reaction. All RPA reactions were stored at -20°C until use.

[0073] Example 7

[0074] Non-malleioid Burkholderia clinical isolates were purchased from the diagnostic laboratory of Maharaj Nakorn Hospital, Chiang Mai. Colonies were scraped directly from sheep blood agar plates and resuspended in 200 μl of water. Genomic DNA was extracted using the GeneJet Genomic DNA Purification Kit (ThermoFisher Scientific, USA, #K0721) according to the manufacturer's protocol. Burkholderia pseudomallei clinical isolates were obtained from 33 patients with melioidosis admitted to Sunpasitthiprasong Hospital, Ubon Ratchathani, Thailand. Samples were obtained from blood, urine, or throat swabs of patients with melioidosis and cultured on Ashdown agar to select for the growth of Burkholderia pseudomallei. For each sample, one colony was picked and streaked on Columbia agar and incubated overnight at 37°C. A loopfull of B. pseudomallei from Columbia agar was then inoculated in 3 mL of Luria-Bertani liquid medium and incubated for 16-18 hours at 37°C with shaking at 200 rpm in a biosafety level 3 laboratory. B. pseudomallei was harvested and lysed for genomic DNA extraction using the QIAmp Mini kit protocol (QIAGEN, Germany, #51304).

[0075] Example 8

[0076] CRISPR reaction was performed in a final 100 μl volume, which contained 100 nM crRNA, 200 nM MBP-LbCas12a, 500 nM FAM-quencher probe and 1x CR buffer 1 (10 Tris-HCl pH 8.0, 50 mM NaCl, 13 mMMgCl2, 1% v / v glycerol). First, CRISPR-Cas12a ribonucleoprotein (RNP) was preassembled into a 30 μl volume with crRNA, MBP-LbCas12a and CR buffer 1. The reactants were incubated at room temperature for 15 minutes. 70 μl FAM-quencher probes were added to RNPs with different amounts of RPA mixtures. The CRISPR reaction was transferred to a fluorescence plate reader Synergy H4 (BioTek, USA) and read at 483 / 530 nm (excitation / emission) every 3 minutes at 37 ° C for 3 hours.

[0077] To verify that the identified target sites can be used with CRISPR-Cas12a to detect Burkholderia pseudomallei genomic DNA, the present disclosure designed CRISPR-Cas12a to include target sites in the crRNA spacer and applied these modified CRISPR-Cas12a enzymes to the DETECTR assay ( Figure 2A ). In the DETECTR assay, pathogen DNA is pre-amplified by an isothermal method and then combined with pre-assembled CRISPR-Cas12a RNP in a CRISPR reaction

[29] . The reaction contains an excess of short ssDNA probes modified with fluorescein and a quencher at the 5' and 3' ends, respectively (FAM-quencher probes). In the presence of pathogen DNA, the programmed CRISPR-Cas12a RNP complex recognizes the intended target and activates itself to form a non-specific ssDNA enzyme that cleaves the probe in trans. The release of fluorescein from the quencher results in fluorescence emission, where fluorescence is a proxy for the detection of pathogen DNA.

[0078] The present disclosure designed 35-nt primers and performed RPA at only 7 target sites. crBP33 was an exception, as it was not possible to design optimal primers due to the highly repetitive sequence and GC-rich nature of this region. The results showed that RPA of target sites crBP34, crBP36, and crBP38 produced good amplification products in 7 target sites. Therefore, the present disclosure selected these candidate target sites and designed their 20-nt sequences (excluding PAM) into the spacer region ( Figure 1A ). A 7-nt RNA extension was also added to the 3' end of the crRNA as it has been shown to increase collateral cleavage activity

[43] . Genomic DNA of 11 pathogens that are common causes of bacterial infection in areas where melioidosis is endemic were preamplified using RPA and each crRNA was tested for each pathogen. These pathogens include Streptococcus pyogenes, Streptococcus epidermidis, Klebsiella pneumoniae, Streptococcus agalactiae, Pseudomonas aeruginosa, Burkholderia thailandensis, Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, and Burkholderia pseudomallei. Fluorescence activation results showed that all three crRNAs could specifically detect Burkholderia pseudomallei, but not the other pathogens ( Figure 2B , 2C and 2D). Encouragingly, all three crRNAs can effectively distinguish Burkholderia thailandensis, a close relative of Burkholderia pseudomallei. This specificity exceeded expectations, as the fluorescence signal from non-Thai Burkholderia was only slightly above background levels, while in contrast, the intensity of Burkholderia pseudomallei was 4 orders of magnitude. Since it is possible to use this platform to diagnose human melioidosis, the present disclosure also tested crBP34, 36 and 38 with human genomic DNA. The inventors found that all three crRNAs did not cross-react with human DNA ( Figure 2B , 2C and 2D). These results validate the crRNA design pipeline that excludes cross-reactivity with non-Thai Burkholderia DNA ( Figure 1A ). In addition, the present disclosure notes that each crRNA exhibits different detection kinetics. The crBP34 signal is the strongest, reaching a maximum at 24 minutes, while crBP36 reaches a maximum at 48 minutes. On the other hand, crBP38 shows a delayed maximum at 108 minutes. These changes may be the result of cumulative differences in RPA efficiency and the intrinsic properties of each crRNA. In any case, all three crRNAs showed specific signals above background in less than 10 minutes after the start of the reaction. Because crBP34 has the strongest signal and the fastest kinetics, the inventors chose crBP34 for the subsequent experiments detailed in Example 9.

[0079] Example 9

[0080] RNP complex assembly and CRISPR reaction were combined and performed in a final 50 μl volume, containing 1xCR buffer 1, 100 nM crRNA, 200 nM MBP-LbCas12a, 100 nM FAM-biotin probe and 5 μl RPA. The reaction was incubated at 37 ° C for 20 minutes. The Hybri-Detect lateral flow test ruler (Milenia Biotek, Germany, #MGHD1) was directly immersed in the reaction and developed for 2-5 minutes.

[0081] Real-time PCR primers have been reported previously

[17] . Primers were synthesized and desalted by Macrogen (Korea). 20 μl real-time PCR reactions were prepared using Maxima SYBR Green / ROX qPCR Master Mix (ThermoFisherScientific, USA, #K0221) according to the manufacturer's instructions. Real-time PCR analysis was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems) under the following conditions: 95°C (10 min); 40 cycles of 95°C (15 s), 61°C (30 s), and 72°C (30 s). Standard melting curve analysis was also performed at the end of PCR. The ΔRn threshold was automatically calculated by the built-in program.

[0082] An important feature of a reliable diagnostic tool is sensitivity. In order to study the ability of crBP34-coupled DETECTR to detect trace amounts of genetic material often present in clinical samples, the present disclosure determines the limit of detection (LOD) of the CRISPR platform. As described above, the known copy number of Burkholderia pseudomallei genomic DNA was serially diluted and used for the assay. The results showed that the LOD of the developed CRISPR-Cas12a detection platform was 40 copies of Burkholderia pseudomallei genomic DNA per reaction ( Figure 3A ). In fact, the assay could detect as few as 4 replicates of input genomic DNA; however, at this lower DNA amount, the detection results were inconsistent, the fluorescence signal was weak, and there was a large detection delay rate (maximum of 55 minutes). For comparison, the present disclosure also performed real-time PCR, a sensitive standardized method for nucleic acid detection. When the LOD of crBP34-DETECTR was compared with the LOD of real-time PCR, it was found that the developed detection platform performed the same as this standard method, which showed reliable detection of 40 replicates per reaction ( Figure 3B ). In addition, the inventors tested the detection platform with Burkholderia pseudomallei genomic DNA spiked with different amounts of human genomic DNA. As expected, the results showed that the presence of human DNA background had a negative impact on sensitivity, especially at the LOD of the assay.

[0083] To eliminate the requirement for an expensive laboratory setup, the inventors of the present disclosure replaced fluorescence detection with a lateral flow dipstick for the assay readout. Thus, in the CRISPR reaction, the FAM-Quencher ssDNA probe was replaced with a FAM-biotin ssDNA probe. The sample pad of the lateral flow dipstick was then dipped directly into the CRISPR reaction. The sample pad contained gold-conjugated anti-FAM antibodies that labeled the FAM moiety for visualization—either in the intact probe or in the cleaved probe ( Figure 3C ). In the absence of B. pseudomallei target DNA, the probes are intact and captured on the streptavidin line via biotin-streptavidin interactions. However, in the presence of target DNA, the incidental activity of CRISPR-Cas12a degrades the probes, releasing the FAM-antibody complexes from the biotin so that they can diffuse further on the dipstick to bind the anti-IgG line. The present disclosure tested this readout with varying amounts of B. pseudomallei genomic DNA and found that the sensitivity of this readout correlated closely with that of the fluorescence readout, with an LOD of 40 replicates per reaction ( Figure 3D ). Therefore, lateral flow dipsticks can be used to provide a sensitive assay readout, offering a simple method for detecting Burkholderia pseudomallei.

[0084] Computer analysis showed that all eight identified CRISPR-Cas12a target sites were highly conserved. The sequence of crBP34 matched 3,335 of 3,341 Burkholderia pseudomallei genomes (Table 1) and appeared to be very specific in preliminary data ( Figure 2B ). To verify that the currently designed crBP34 can specifically detect other Burkholderia pseudomallei isolates, the present disclosure tested the developed CRISPR diagnostic platform with various clinically isolated bacteria, in particular Burkholderia pseudomallei and common non-Burkholderia pseudomallei pathogens. The results showed that crBP34 can accurately detect all Burkholderia pseudomallei isolates (n=33) ( Figure 4 ). Importantly, the assay distinguished all other DNA from non-B. pseudomallei pathogens (n=88). The fluorescent signal from the latter group of pathogens remained at baseline, at the same level as the background (water input RPA, "none"). In summary, these data indicate that crBP34 exhibits 100% specificity and has the potential to be used to detect B. pseudomallei from different geographic regions. Table 2 below further illustrates the descriptive statistics obtained for various clinical bacterial isolates tested using the developed crBP34-DETECTR (or SEQ ID No. 3-DETECTR).

[0085] Table 2

[0086]

[0087] Example 10

[0088] Nucleic acid detection by RPA and LAMP isothermal amplification assays have been increasingly used in the research community due to their high sensitivity and simplicity. However, LAMP requires a series of 6 to 8 primers to work together at optimal sites

[44] . This requirement may limit its widespread use in the detection of Burkholderia pseudomallei, whose genome is heterogeneous and GC-rich. On the other hand, RPA can tolerate significant mismatches in primers

[32] , making it less specific. The inventors often observed nonspecific amplification in the RPA reactions they performed. In contrast, the DETECTR assay combines RPA and CRISPR-Cas12a. It utilizes CRISPR-Cas12a as a sequence-specific DNA reader to verify the identity of the pre-amplified DNA in the RPA reaction. This adds additional stringency to RPA while maintaining its high sensitivity. Therefore, the developed method or assay is highly specific and sensitive. The assay has an LOD of 40 copies per reaction, which is comparable to real-time PCR ( Figure 3B ), LAMP

[26] and RPA[29,30].

[0089] The specificity of crBP34-DETECTR may come from the designed crRNA, crBP34, which is the result of large-scale phylogenetic and bioinformatic analyses to eliminate cross-reactivity ( Figure 1B and 1C ). This is in stark contrast to previously reported detection methods using real-time PCR[21-25], LAMP[26-27], or RPA[28-30], for which primers were designed based on selected genomic loci or limited bioinformatics analysis of a few reference strains. Testing crBP34 and other crRNAs with Burkholderia pseudomallei isolates from other geographic regions requires further examination to assess their general applicability. However, coverage analysis predicted that both the CRISPR-Cas12 target site (Table 1) and the RPA primers are present in more than 97% of the global Burkholderia pseudomallei population. This gives a high probability of detection by the designed crRNA.

[0090] When human DNA was mixed with B. pseudomallei DNA, the inventors observed a reduction in RPA products, leading to a subsequent loss of collateral activity of CRISPR-Cas12a. Therefore, DNA extraction methods that selectively enrich for pathogens from clinical samples will play a key role in detection.

[0091] Aspects of specific embodiments of the present disclosure address at least one aspect, problem, limitation and / or disadvantage of existing kits or methods associated with rapid and relatively easy detection of Burkholderia pseudomallei in biological samples obtained from human subjects. Although features, aspects and / or advantages associated with certain embodiments have been described in the present disclosure, other embodiments may also exhibit these features, aspects and / or advantages, and not all embodiments must exhibit these features, aspects and / or advantages to fall within the scope of the present disclosure. Those of ordinary skill in the art will understand that the several structures, components or alternatives thereof disclosed above may be ideally combined into alternative structures, components and / or applications. In addition, within the scope of the present disclosure, various modifications, changes and / or improvements may be made to the various embodiments disclosed by those of ordinary skill in the art, which are only limited by the following claims.

[0092] References:

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Claims

1. A CRISPR RNA (crRNA) adapted for use in a CRISPR-based ribonucleoprotein (RNP) system for responding to one or more genetic materials derived from Burkholderia pseudomallei in a biological sample, comprising: a spacer region, the spacer region having one of the polynucleotide sequences selected from SEQ ID 3, SEQ ID 5 and SEQ ID 7; A repeat region preceding the spacer region, forming its secondary structure; and A first extension region is disposed immediately after the spacing region.

2. The crRNA according to claim 1, characterized in that The spacer is configured to hybridize to a complementary sequence residing in the genetic material, and the complementary sequence is preceded by a pre-spacer adjacent motif (PAM) comprising a TTTV sequence, wherein V is a non-T nucleotide.

3. The crRNA according to claim 1, characterized in that The invention also comprises a second extension region, wherein the second extension region has a Gs sequence, and a GGG sequence is arranged in front of the repeating region.

4. The crRNA according to claim 1, characterized in that The repeat region comprises a polynucleotide sequence of SEQ ID No.9 or SEQ ID No.

10.

5. The crRNA according to claim 1, characterized in that The first stretch region includes a polynucleotide sequence of AAAGGAA.

6. The crRNA according to claim 1, characterized in that The CRISPR-based RNP system is a CRISPR-Cas12a RNP system.

7. A method for detecting the presence of one or more genetic materials of Burkholderia pseudomallei obtained from a biological sample, comprising the steps of: providing one or more genetic materials of Burkholderia pseudomallei; Assembling a CRISPR-based RNP system, the assembled CRISPR-based RNP system comprising a crRNA, wherein the spacer region thereof has one of the polynucleotide sequences selected from SEQ ID No. 3, SEQ ID No. 5, and SEQ ID No. 7; reacting the genetic material with an assembled CRISPR-based RNP system along with a plurality of reporter probes in a buffer solution, the CRISPR-based RNP system being configured to activate side branch cleavage of the reporter probes to generate a signal upon hybridization to a polynucleotide sequence complementary to a spacer region located within the one or more genetic materials; and The emitted signal is detected to confirm the presence of Burkholderia pseudomallei in the biological sample.

8. The method according to claim 7, characterized in that The spacer is configured to hybridize to a complementary sequence residing in the genetic material, and the complementary sequence is preceded by a pre-spacer adjacent motif (PAM) comprising a TTTV sequence, wherein V is a non-T nucleotide.

9. The method according to claim 7, characterized in that: The detection step further comprises subjecting a portion of the reacted buffer solution to a lateral flow assay to capture the emitted signal.

10. The method according to claim 7, characterized in that The crRNA also includes a repeating region before the spacer region that forms its secondary structure, and the repeating region includes a polynucleotide sequence of SEQ ID No.9 or SEQ ID No.

10.

11. The method according to claim 7, characterized in that The crRNA also includes a first extension region arranged immediately after the spacer region, and the first extension region includes a polynucleotide sequence of AAAGGAA.

12. The method according to claim 7, characterized in that The crRNA also includes a second extension region having a GGG sequence arranged before the repeat region.

13. The method according to claim 7, characterized in that The CRISPR-based RNP system is a CRISPR-Cas12a RNP system.

Images