Integration of the Ligase Chain Reaction with the CRISPR-Cas12a System for Homogeneous, Ultrasensitive, and Visual Detection of microRNA.

The ligase chain reaction (LCR), as a classic nucleic acid amplification technique, is popular in the detection of DNA and RNA due to its simplicity, powerfulness, and high specificity. However, homogeneous and ultrasensitive LCR detection is still quite challenging. Herein, we integrate the LCR with a CRISPR-Cas12a system to greatly promote the application of the LCR in a homogeneous fashion. By employing microRNA as the model target, we design LCR probes with specific protospacer adjacent motif sequences and the guide RNA. Then, the LCR is initiated by target microRNA, and the LCR products specifically bind to the guide RNA to activate the Cas12a system, triggering secondary signal amplification to achieve ultrasensitive detection of microRNA without separation steps. Moreover, by virtue of a cationic conjugated polymer, microRNA can not only be visually detected by naked eyes but also be accurately quantified based on RGB ratio analysis of images with no need of sophisticated instruments. The method can quantify microRNA up to 4 orders of magnitude, and the determination limit is 0.4 aM, which is better than those of other reported studies using CRISPR-Cas12a and can be compared with that of the reverse-transcription polymerase chain reaction. This study demonstrates that the CRISPR-Cas12a system can greatly expand the application of the LCR for the homogeneous, ultrasensitive, and visual detection of microRNA, showing great potential in efficient nucleic acid detection and in vitro diagnosis.

Pyrococcus furiosus Argonaute coupled with modified ligase chain reaction for detection of SARS-CoV-2 and HPV.

Infectious diseases caused by viruses such as SARS-CoV-2 and HPV have greatly endangered human health. The nucleic acid detection is essential for the early diagnosis of diseases. Here, we propose a method called PLCR (PfAgo coupled with modified Ligase Chain Reaction for nucleic acid detection) which utilizes PfAgo to only use DNA guides longer than 14-mer to specifically cleave DNA and LCR to precisely distinguish single-base mismatch. PLCR can detect DNA or RNA without PCR at attomolar sensitivities, distinguish single base mutation between the genome of wild type SARS-CoV-2 and its mutant spike D614G, effectively distinguish the novel coronavirus from other coronaviruses and finally achieve multiplexed detection in 70 min. Additionally, LCR products can be directly used as DNA guides without additional input guides to simplify primer design. With desirable sensitivity, specificity and simplicity, the method can be extended for detecting other pathogenic microorganisms.

Integration of magnetic separation and real-time ligation chain reaction for detection of uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) is a protein enzyme that initiates the base excision repair pathway for maintaining genome stability. Sensitive detection of UDG activity is important in the study of many biochemical processes and clinical applications. Here, a method for detecting UDG is proposed by integrating magnetic separation and real-time ligation chain reaction (LCR). First, a DNA substrate containing uracil base is designed to be conjugated to the magnetic beads. By introducing a DNA complementary to the DNA substrate, the uracil base is recognized and removed by UDG to form an apurinic/apyrimidinic (AP) site. The DNA substrate is then cut off from the AP site by endonuclease IV, releasing a single-strand DNA (ssDNA). After magnetic separation, the ssDNA is retained in the supernatant and then detected by real-time LCR. The linear range of the method is 5 × 10-4 to 5 U/mL with four orders of magnitude, and the detection limit is 2.7 × 10-4 U/mL. In the assay, ssDNA template obtained through magnetic separation can prevent other DNA from affecting the subsequent LCR amplification reaction, which provides a simple, sensitive, specific, and universal way to detect UDG and other repair enzymes. Furthermore, the real-time LCR enables the amplification reaction and fluorescence detection simultaneously, which simplifies the operation, avoids post-contamination, and widens the dynamic range. Therefore, the integration of magnetic separation and real-time LCR opens a new avenue for the detection of UDG and other DNA repair enzymes.

Development of a Polymerase Chain Reaction/Ligase Detection Reaction Assay for Detection of CYP2C19 Polymorphisms.

AIMS: Cytochrome P450 2C19 (CYP2C19) genotypes are associated with differential drug metabolism. The aim of this study was to establish a reliable assay for CYP2C19 genotyping based on a polymerase chain reaction/ligase detection reaction (PCR-LDR). MATERIALS AND METHODS: Specific primers and probes were designed to detect CYP2C19*1, *2, *3, and *17. A control for each allele was prepared and used for performance evaluation. A total of 200 clinical samples were analyzed using the PCR-LDR assay and Sanger sequencing. RESULTS: The detection limit of the PCR-LDR assay was 2 ng/µL of genomic DNA. Common interfering substances in the blood did not affect the results of the detection. For the clinical samples, the results of the PCR-LDR and the Sanger sequencing were identical. Among the 200 patients, 104 (52%) were wild type (*1/*1), 64 (32%) were *1/*2, 16 (8%) were *1/*3, 8 (4%) were *2/*2, 7 (3.5%) were *2/*3, and 1 (0.5%) was *1/*7. No *3/*3 genotype was detected in these patients. CONCLUSION: This PCR-LDR assay is reliable for the detection of CYP2C19 genotypes in a clinical setting. It will be a useful tool to screen for CYP2C19 loss-of-function alleles in patients before clopidogrel and proton pump inhibitor treatment.

Detection and Identification of Uncapped RNA by Ligation-Mediated Reverse Transcription Polymerase Chain Reaction.

The 5'-cap structure is an essential feature in eukaryotic mRNA required for mRNA stability and enhancement of translation. Ceratin transcripts are selectively silenced by decapping in the cytoplasm and later become translationally active again by acquiring the cap structure to regenerate translatable mRNAs. Identification of uncapped mRNA transcripts will reveal how gene expression is regulated by the mRNA recapping pathway. What follows is a sensitive method to detect and identify the uncapped mRNA from the cells. The technique consists of three parts: selective ligation of anchor RNA to the 5'-end of monophosphate RNA by double-strand RNA ligase, conversion of ligated RNA product into cDNA by reverse transcription, and amplification of a specific cDNA by polymerase chain reaction.

Detection of Extracellular Vesicles Using Proximity Ligation Assay with Flow Cytometry Readout-ExoPLA.

Extracellular vesicles (EVs) are continuously released by most cells, and they carry surface markers of their cells of origin. Found in all body fluids, EVs function as conveyers of cellular information, and evidence implicates them as markers of disease. These characteristics make EVs attractive diagnostic targets. However, detection and characterization of EVs is challenging due to their small size. We've established a method, called ExoPLA, that allows individual EVs to be detected and characterized at high specificity and sensitivity. Based on the in situ proximity ligation assay (in situ PLA), proximal oligonucleotide-conjugated antibodies bound to their targets on the surfaces of the EVs allow formation of circular products that can be fluorescently labeled by rolling circle amplification. The intense fluorescent signals produced in this assay allow detection and enumeration of individual EVs by flow cytometry. We describe the procedures for ExoPLA, along with expected results and troubleshooting. © 2017 by John Wiley & Sons, Inc.

Molecular Subtyping of Salmonella Typhimurium with Multiplex Oligonucleotide Ligation-PCR (MOL-PCR).

A multiplex oligonucleotide ligation-PCR (MOL-PCR) assay is a valuable high-throughput technique for the detection of bacteria and viruses, for characterization of pathogens and for diagnosis of genetic diseases, as it allows one to combine different types of molecular markers in a high-throughput multiplex assay. A MOL-PCR assay starts with a multiplex oligonucleotide ligation reaction for detection of the molecular marker, followed by a singleplex PCR for signal amplification and analysis of the MOL-PCR products on a Luminex platform. This last step occurs through a liquid bead suspension array in which the MOL-PCR products are hybridized to MagPlex-TAG beads.In this chapter, we describe the complete procedure for a MOL-PCR assay for subtyping of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant S. 1,4[5],12:i:- from DNA isolation through heat lysis up to data interpretation through a Gödel Prime Product. The subtyping assay consists of 50 discriminative molecular markers and two internal positive control markers divided over three MOL-PCR assays.

Ligation-mediated PCR with a back-to-back adapter reduces amplification bias resulting from variations in GC content.

Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence. The use of this adapter in LM-PCR reduced the impairment of PCR by substrate DNA with a high GC content, compared with the use of traditional LM-PCR adapters. This result suggested that our method has the potential to contribute to reduction of the amplification bias that is caused by an intrinsic property of the sequence context in substrate DNA. A DNA preparation obtained from a chromatin immunoprecipitation assay using pulldown of a specific form of histone H3 was successfully amplified using the modified LM-PCR, and the amplified products could be used as probes in a fluorescence in situ hybridization analysis.

A copy number variation genotyping method for aneuploidy detection in spontaneous abortion specimens.

OBJECTIVE: Chromosomal abnormalities such as aneuploidy have been shown to be responsible for causing spontaneous abortion. Genetic evaluation of abortions is currently underperformed. Screening for aneuploidy in the products of conception can help determine the etiology. We designed a high-throughput ligation-dependent probe amplification (HLPA) assay to examine aneuploidy of 24 chromosomes in miscarriage tissues and aimed to validate the performance of this technique. METHODS: We carried out aneuploidy screening in 98 fetal tissue samples collected from female subjects with singleton pregnancies who experienced spontaneous abortion. The mean maternal age was 31.6 years (range: 24-43), and the mean gestational age was 10.2 weeks (range: 4.6-14.1). HLPA was performed in parallel with array comparative genomic hybridization, which is the gold standard for aneuploidy detection in clinical practices. The results from the two platforms were compared. RESULTS: Forty-nine out of ninety-eight samples were found to be aneuploid. HLPA showed concordance with array comparative genomic hybridization in diagnosing aneuploidy. CONCLUSION: High-throughput ligation-dependent probe amplification is a rapid and accurate method for aneuploidy detection. It can be used as a cost-effective screening procedure in clinical spontaneous abortions. © 2016 John Wiley & Sons, Ltd.

Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer.

We previously developed a separation-free ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye. This assay could successfully detect one cancer mutation among 10 wild-type templates. In the current study, the mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5) that was spectrally similar but more fluorescent.

[The applications of thermostable ligase chain reaction in facilitating DNA recombination].

The traditional Type Ⅱ restriction enzyme-based method is restricted by the purification steps, and therefore, cannot be applied to specific DNA assembly in chaotic system. To solve this problem, Thermostable Ligase Chain Reaction (TLCR) was introduced in the process of DNA assembly and capture. This technique combines the feature of thermostable DNA ligase and sequence specific oligo ligation template, "Helper", to achieve specific assembly of target fragments and exponential increase of products in multiple thermocyclings. Two plasmid construction experiments were carried out in order to test the feasibility and practical performance of TLCR. One was that, TLCR was used to specifically capture a 1.5 kb fragment into vector from an unpurified chaotic system which contained 7 different sizes of fragments. The results showed that the capturing accuracy was around 80%, which proved the feasibility and accuracy of using TLCR to specific assembly of DNA fragments in a complicated mixed system. In the other experiment, TLCR was used to capture two fragments (total length was 27 kb) from Hind Ⅲ digestion of Lambda genome into vector by order. The results also showed an accuracy of around 80%. As demonstrated in the results, TLCR can simplify the process of DNA recombination experiments and is suitable for the assembly of multiple and large DNA fragments. This technique can provide convenience to biological experiments.

Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation.

Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories.

A simple microfluidic assay for the detection of ligation product.

We present a novel microfluidic-based approach to detect ligation products. The conformal specificity of ligases is used in various molecular assays to detect point mutations. Traditional methods of detecting ligation products include denaturing gel electrophoresis, sequence amplification, and melting curve analysis. Gel electrophoresis is a labor- and time-intensive process, while sequence amplification and melting curve analysis require instruments capable of accurate thermal ramping and sensitive optical detection. Microfluidics has been widely applied in genomics, proteomics, and cell cytometry to enable rapid and automated assays. We designed an assay that fluorogenically detects ligation products following a simple magnetic separation through a microfluidic channel. 100 nM of synthetic HIV-1 K103N minority mutant templates were successfully detected in 30 min. This simple and rapid method can be coupled with any ligation assay for the detection of ligation products.

Copy number variation analysis by ligation-dependent PCR based on magnetic nanoparticles and chemiluminescence.

A novel system for copy number variation (CNV) analysis was developed in the present study using a combination of magnetic separation and chemiluminescence (CL) detection technique. The amino-modified probes were firstly immobilized onto carboxylated magnetic nanoparticles (MNPs) and then hybridized with biotin-dUTP products, followed by amplification with ligation-dependent polymerase chain reaction (PCR). After streptavidin-modified alkaline phosphatase (STV-AP) bonding and magnetic separation, the CL signals were then detected. Results showed that the quantification of PCR products could be reflected by CL signal values. Under optimum conditions, the CL system was characterized for quantitative analysis and the CL intensity exhibited a linear correlation with logarithm of the target concentration. To validate the methodology, copy numbers of six genes from the human genome were detected. To compare the detection accuracy, multiplex ligation-dependent probe amplification (MLPA) and MNPs-CL detection were performed. Overall, there were two discrepancies by MLPA analysis, while only one by MNPs-CL detection. This research demonstrated that the novel MNPs-CL system is a useful analytical tool which shows simple, sensitive, and specific characters which are suitable for CNV analysis. Moreover, this system should be improved further and its application in the genome variation detection of various diseases is currently under further investigation.

Oligonucleotide ligation assay detects HIV drug resistance associated with virologic failure among antiretroviral-naive adults in Kenya.

BACKGROUND: Transmitted drug resistance (TDR) is increasing in some areas of Africa. Detection of TDR may predict virologic failure of first-line nonnucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART). We evaluated the utility of a relatively inexpensive oligonucleotide ligation assay (OLA) to detect clinically relevant TDR at the time of ART initiation. METHODS: Pre-ART plasmas from ART-naive Kenyans initiating an NNRTI-based fixed-dose combination ART in a randomized adherence trial conducted in 2006 were retrospectively analyzed by OLA for mutations conferring resistance to NNRTI (K103N, Y181C, and G190A) and lamivudine (M184V). Post-ART plasmas were analyzed for virologic failure (≥1000 copies/mL) at 6-month intervals over 18-month follow-up. Pre-ART plasmas of those with virologic failure were evaluated for drug resistance by consensus and 454-pyrosequencing. RESULTS: Among 386 participants, TDR was detected by OLA in 3.89% (95% confidence interval: 2.19 to 6.33) and was associated with a 10-fold higher rate of virologic failure (hazard ratio: 10.39; 95% confidence interval: 3.23 to 32.41; P < 0.001) compared with those without TDR. OLA detected 24 TDR mutations (K103N: n = 13; Y181C: n = 5; G190A: n = 3; M184V: n = 3) in 15 subjects (NNRTI: n = 15; 3TC: n = 3). Among 51 participants who developed virologic failure, consensus sequencing did not detect additional TDR mutations conferring high-level resistance, and pyrosequencing only detected additional mutations at frequencies <2%. Mutant frequencies <2% at ART initiation were significantly less likely to be found at the time of virologic failure compared with frequencies ≥2% (22% vs. 63%; P < 0.001). CONCLUSIONS: Detection of TDR by a point mutation assay may prevent the use of suboptimal ART.

A label-free and PCR-free electrochemical assay for multiplexed microRNA profiles by ligase chain reaction coupling with quantum dots barcodes.

The profiling of microRNAs (miRNAs) is greatly significant for cellular events or disease diagnosis. Electrochemical methods for miRNAs analysis mostly can only measure one kind of miRNA, which is unambiguous to indicate the disease type and state. Here a label-free and PCR-free electrochemical method is presented for multiplexed evaluation of miRNAs in a single-tube experiment. The method is based on the combination of the high base-mismatch selectivity of ligase chain reaction (LCR) and the remarkable voltammetric signature of electrochemical QDs barcodes. Two reporting probes of RP1 and RP2 were labeled with PbS and CdS quantum dots (QDs) to prepare PbS-RP1 and CdS-RP2 conjugates, and two capture probes of CP1 and CP2 were co-immobilized on magnetic beads (MBs) to fabricate MB-CP1CP2 conjugate. The miRNAs samples were simply incubated with MB-CP1CP2, PbS-RP1, and CdS-RP2 conjugates, and then added with T4 DNA ligase. After release of the disjoined QDs barcodes from the MB-conjugates, two target miRNAs of miR-155 and miR-27b were simultaneously detected by square wave voltammetry with linear ranges of 50 fM-30 pM and 50 fM-1050 pM, and limits of detection (LODs) of 12 fM and 31 fM (S/N=3). The method fulfilled the assay in less than 70 min, and showed acceptable testing recoveries for the determination of miRNAs in biological matrix. Currently there are rare reports about electrochemical multiplexed quantification of miRNAs. The method is likely to provide a new platform for identification of multiple miRNAs in a simple way.

Highly sensitive and specific multiplexed microRNA quantification using size-coded ligation chain reaction.

As important regulators of gene expression, microRNAs (miRNAs) are emerging as novel biomarkers with powerful predictive value in diagnosis and prognosis for several diseases, especially for cancers. There is a great demand for flexible multiplexed miRNA quantification methods that can quantify very low levels of miRNA targets with high specificity. For further analysis of miRNA signatures in biological samples, we describe here a highly sensitive and specific method to detect multiple miRNAs simultaneously in total RNA. First, we rationally design one of the DNA probes modified with two ribonucleotides, which can greatly improve the ligation efficiency of DNA probes templated by miRNAs. With the modified DNA probes, the ligation chain reaction (LCR) can be well applied to miRNA detection and as low as 0.2 fM miRNA can be accurately determined. High specificity to clearly discriminate a single nucleotide difference among miRNA sequences can also be achieved. By simply coding the DNA probes with different length of oligo (dA) for different miRNA targets, multiple miRNAs can be simultaneously detected in one LCR reaction. In our proof of principle work, we detect three miRNAs: let-7a, mir-92a, and mir-143, which can also be simultaneously detected in as small as 2 ng of total RNA sample.

Ligase chain reaction amplification for sensitive electrochemiluminescent detection of single nucleotide polymorphisms.

Single nucleotide polymorphisms are the most common type of genetic variations among human beings and can serve as biomarkers for various types of diseases. In this work, based on ligase chain reaction amplification for the production of massive hemin/G-quadruplex DNAzymes to quench the electrochemiluminescent (ECL) emission of quantum dots (QDs), a universal and sensitive single nucleotide polymorphism detection method is described. During the ligase chain reaction process, the mutant K-ras target gene is recycled and exponentially duplicated, leading to the attachment of numerous G-rich sequences on the QD-embedded sensing surface. Upon the addition of the assistant sequences and hemin, numerous hemin/G-quadruplex DNAzymes are formed, which consume the dissolved oxygen in the detection buffer and result in significant quenching of QD ECL emission for sensitive single nucleotide polymorphism determination. The developed method shows a linear range of 50 fM to 50 pM and an estimated detection limit of 45 fM for the mutant K-ras gene. The proposed strategy also exhibits high selectivity towards the mutant K-ras gene against the co-existence of 10(3)-fold excess of the wild-type K-ras gene, which makes our method a useful addition to the alternatives for single nucleotide polymorphism monitoring.