Vis-Fusion LIEXA: A Point-of-Care Testing Method for Highly Specific

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Vis-Fusion LIEXA: A Point-of-Care Testing Method for Highly Specific and Sensitive Quantitation of Fusion Gene with SmartPhone Hui Wang, Honghong Wang, Yuting Jia, Ruyan Sun, Weixiang Hong, Mai Zhang, and Zhengping Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b03061 • Publication Date (Web): 29 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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Analytical Chemistry

Vis-Fusion LIEXA: A Point-of-Care Testing Method for Highly Specific and Sensitive Quantitation of Fusion Gene with SmartPhone Hui Wang, Honghong Wang, Yuting Jia, Ruyan Sun, Weixiang Hong, Mai Zhang, and Zhengping Li* School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing 100083 P. R. China. ABSTRACT: Fusion genes, playing a causal role in human tumorigenesis and developments, are deemed as gold standard molecular biomarkers in cancer diagnosis, therapy, and prognosis. A rapid, robust and sensitive method detection of fusion gene for point-ofcare (POC) diagnosis is urgently needed. Here, taking the advantages of the superior specificity of ligation reaction and highly amplified efficiency of isothermal exponential amplification with pH indicator, we developed a colorimetric method for visual detection of fusion gene with high sensitivity and specificity by the naked eye. More importantly, we first found that fusion gene can be accurately quantified in a wide dynamic range (2 zmol to 2 fmol) by an open-source App with SmartPhone-assisted RGB (Red, Green, and Blue value) reading mode. The proposed method for Visual detection of Fusion gene by Ligation-triggered Isothermal Exponential Amplification is termed Vis-Fusion LIEXA. We have demonstrated that the Vis-Fusion LIEXA is a practical and reliable method for accurate quantitative detection of the fusion gene in a complex biological sample at zmol level in 40 min only with a SmartPhone, thereby providing a user-friendly and point-of-care testing (POCT) tool for molecular diagnostics.

Fusion genes, also called chimeric genes or hybrid genes, are chimeras of two partner genes originated from chromosomal structural rearrangements such as translocations, deletions, or inversions. The resulting protein products may lead to the abnormal status of expression levels, functions and action sites, which in return may cause the abnormal proliferation of cells and tumorigenesis.1, 2 Fusion gene transcripts are excellent tumor-specific biomarkers providing a new view of tumor pathogenesis and early cancer diagnosis due to they are present in tumor tissue and absent in healthy tissue.3 For instance, the Philadelphia chromosome translocation (t(q;22) (q34;q11)) results in the molecular juxtaposition of BCR and ABL genes, to form the well-known BCR-ABL fusion gene. The fusion transcript codes the chimeric BCR-ABL protein which has constitutively elevated tyrosine phosphokinase activity leading to chronic myeloid leukemia (CML).4 As a drug target, fusion genes have impressive therapeutic significance, such as the first success of Imatinib for treating BCR-ABL fusion gene-caused CML and the recent victory of Crizotinib and/or Ceritinib for treating ALK-rearranged fusion gene-driven non-small-cell lung cancer (NSCLC).5-8 With the widespread applications of high-throughput next-generation DNA/RNA sequencing technologies and persistent efforts in bioinformatics, many new cancer-associated fusion genes have been discovered.9-12 Since the significant role of the fusion gene in human cancers, a specialized database (Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer) has been created13. More and more fusion genes are used as gold standard molecular biomarkers by National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for cancers diagnosis, therapy, and prognosis. Up to now, the fusion gene analysis methods mainly divided to two categories: (1) transcriptome-wide screening methods, such as Next Generation Sequencing (NGS)9, DNA array14, 15,

and NanoString nCounter16, 17. These high-throughput technologies are powerful tools for transcriptome-wide screening fusion gene transcripts at an unprecedented level and uncovering the veil between fusion genes and cancers. However, the high-throughput methods mentioned above are seriously limited in ordinary diagnostic laboratories due to their laborious and time-consuming experimental procedures, the high cost of instruments and reagents, the requirement of a large amount of the RNA samples and complicated data analysis. (2) Traditional methods for detecting fusion genes in medical laboratories include chromosome banding analysis (karyotyping), fluorescence in situ hybridization (FISH), and reverse transcription polymerase chain reaction (RT-PCR). Karyotyping requires fresh cells for short-term culturing to obtain metaphase chromosomes is labor intensive and can be technically challenging, with a fairly lengthy runaround time and low success rate in solid tumors.18 For these reasons, karyotyping is rapidly becoming obsolete in the face of technologies advances. FISH with locus-specific probes can be applied for fusion gene analysis in fresh/frozen or FFPE tissue with high specificity and sensitivity. But it is a highly skilled job and time-consuming. Among these, RT-PCR is the most favorite method for quantitatively detecting fusion gene transcripts.20-23 However, the PCR-based methods have shown some inherent limitations, such as the requirements of the stringent design of target-specific primers, precisely optimized experimental conditions, and the false-positive results rising from the cycling amplification artifacts (such as primerdimers).24,25 The specificity of the PCR-based method is generally achieved by using primer pairs targeting each partner gene. Unfortunately, the primer pairs can also amplify other homologous fusion transcripts resulting in undesired nonspecific amplification, which greatly challenge the specificity and sensitivity for PCR-based fusion gene assay. Thus, a Point-of-Care testing (POCT) method for the specific

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identification and highly sensitive detection of fusion genes is urgently needed in the clinical molecular diagnostic assay. To address these challenges, herein, taking the advantages of the superior specificity of ligation reaction and highly amplified efficiency of isothermal exponential amplification, we developed a sensitive and specific method for fusion gene assay. By using a common and cheap pH indicator, fusion gene can be visually detected by the naked eye. More importantly, by coupling SmartPhone-assisted results readout, the fusion gene can be accurately quantified by SmartPhone without other expensive instruments. This user-friendly and POCT method for Visual detection of Fusion gene by Ligation-triggered Isothermal Exponential Amplification is termed Vis-Fusion LIEXA.

EXPERIMENTAL SECTION Materials. Ammonium sulfate ((NH4)2SO4), dithiothreitol (DTT), potassium chloride (KCl), 4S Red Plus Nucleic Acid Stain (10000×, aqueous solution) were supplied by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). Cresol red, 8 M potassium hydroxide solution and 1 M magnesium sulfate solution were purchased from Sigma-Aldrich (Shanghai, China). 10 mM Adenosine 5'-Triphosphate (ATP), SplintR® ligase and high concentration (120 units/L) Bst WarmStart 2.0 DNA polymerase large fragment were obtained from New England Biolabs. 10 mM deoxynucleotide solution mixture (10 mM dATP, 10 mM dTTP, 10 mM dCTP, and 10 mM dGTP), 6× loading buffer, and 20 bp DNA ladder (Dye Plus) were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). All reagents were of analytical grade and were used as received without further purification. RNA and oligonucleotides stock solutions, low buffered Ligation SUPER Mix and low buffered WarmStart Colorimetric IEXA SUPER Mix were prepared with nuclease enzyme-free water (Thermal Fisher Scientific). All oligonucleotides used in this work were synthesized and purified by TaKaRa Biotechnology Co., Ltd. The sequences were listed in Table S1. Preparation of low buffered 2× Ligation SUPER Mix. According to the product information of SplintR ligase and the previous literature26, the 2× Ligation SUPER Mix was prepared as the following components: 20 mM MgCl2, 2 mM ATP, 20 mM DTT, 5 units (25 units/µL in stock) SplintR ligase, and a small amount of Tris-HCl from the ligase storage buffer (10 mM Tris-HCl in the ligase storage buffer, 100 µM in final ligation reaction). All components were mixed in nuclease-free water and the pH of the SUPER Mix was adjusted to ~8.5 with 1 M KOH. The pH was measured by pH-indicator strips (McolorpHastTM pH 5.0-10.0, Merck Millipore). Preparation of low buffered 2× WarmStart Colorimetric IEXA SUPER Mix. The low buffered 2× WarmStart Colorimetric IEXA SUPER Mix was prepared as the following components: 100 mM KCl, 10 mM (NH4)2SO4, 10 mM MgSO4, 5 mM dNTPs, 0.2% Triton X-100, 12 units Bst WarmStart 2.0 DNA polymerase, 200 µM Cresol red, and 100 µM Tris-HCl from the high concentration (120 units/L) Bst WarmStart 2.0 DNA polymerase storage buffer (10 mM Tris-HCl in the storage buffer, 50 µM in final IEXA reaction). All components were mixed in nuclease-free water and the pH of the SUPER Mix was adjusted to ~8.5 with 1 M KOH. The pH was measured by pH-indicator strips.

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The experimental protocols for fusion gene detection by Vis-Fusion LIEXA. (1) Ligation reaction: the ligation reaction mixture consisted of 1× Ligation SUPER Mix (10 mM MgCl2, 1 mM ATP, 10 mM DTT, 2.5 units SplintR ligase (100 nM), and 50 µM Tris-HCl, pH ~8.5 @ 25°C), 200 pM probe SLPe13, 200 pM probe SLP-a2, and appropriate amount of fusion gene sample (including e13a2, or e1a2, or e14a2, or e19a2, or total RNA samples) in a reaction volume of 10 μL. All fusion sample was annealed at 75 °C for 2 min before carrying out the ligation reaction. The reaction mixture was incubated at 25 °C for 20 min to complete the hybridization between the two specific probes and target RNAs, meanwhile, complete the ligation reaction. After the ligation reaction, the products were immediately put on ice. (2) IEXA reaction: the product of ligation reaction was added into IEXA reaction mixture with a final volume of 20 μL. The IEXA reaction mixture consisted of 1× WarmStart Colorimetric IEXA SUPER Mix (50 mM KCl, 5 mM (NH4)2SO4, 5 mM MgSO4, 2.5 mM dNTPs, 0.1% Triton X-100, 6 units Bst WarmStart 2.0 DNA polymerase, 100 µM Cresol red, and 25 µM Tris-HCl, 0.6 μM UP1, 0.6 μM UP2). The reaction mixture was incubated at 65 °C for 16 min to perform IEXA reaction. To facilitate further analysis, 8-tube strips (0.2 mL, Thermal Fisher Scientific) were used in the IEXA step. (3) Results Analysis: after IEXA reaction, the reaction tubes were immediately put on ice for 20 sec and followed at room temperature 10 sec, then the colorimetric results were photographed by a SmartPhone (iPhone 8). Through adjusting the viewing position (black cross), RGB values (Red, Green, and Blue) of each reaction tubes can be quickly obtained by using an open-source image analysis App (Color Picker) on SmartPhone. The RGB values were further analyzed by excel or Origin 8. Cell culture and extraction of total RNA. The HeLa, MCF7, and MRC-5 cells were cultured in DMEM Medium (GBICO) containing 10% (v/v) FBS (GBICO), 100 U/mL penicillin, 100 μg/mL streptomycin and 3 mmol/L L-glutamine at 37 °C in a humidified atmosphere containing 5% CO2. K562 cell was cultured in RPMI 1640 Medium (Sigma) containing 10% (v/v) FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and 3 mmol/L L-glutamine at 37 °C in a humidified atmosphere containing 5% CO2. The total RNA samples were extracted from different cell lines by using the TRIzol® Reagent (Invitrogen) according to the manufacturer’s protocol. The concentration of total RNA was quantified from the absorption at 260 nm with NanoDrop One (Thermo Scientific), and then stored in -80 °C. Non-denaturing Polyacrylamide Gel Electrophoresis (PAGE) Analysis. Non-denaturing polyacrylamide gel electrophoresis (PAGE) was used to confirm the products of LIEXA reaction. The products of LIEXA reaction (5 µL) mixed with 6× loading buffer (1 µL) was loaded on to a 16% nondenaturing PAGE gel. The PAGE experiments were performed in 1× TBE (90 mM Tris, 90 mM Boric acid, 2 mM EDTA, pH=8.3 @ 25 °C) buffer at 120 V for 80 min. Subsequently, the gel was stained by 2× 4S Red Plus Nucleic Acid Stain for 5 min in 1× TBE buffer. Finally, the gel was imaged by using a Gel Doc EZ Imaging System (Bio-Rad, USA).


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Figure 1. The overview of Vis-Fusion LIEXA method for fusion gene assay. (A) BCR and ABL gene with breakpoints. (B) BCR-ABL fusion transcripts. (C) Design of fusion transcript (e13a2 as a model in this work)-specific stem-loop DNA probes (SLP-e13 and SLP-a2). (D) Target-splinted ligation of two stem-loop probes by SplintR ligase and IEXA reaction catalyzed by Bst WarmStart 2.0 DNA polymerase. (E) The process of the IEXA reaction for each dNTP incorporated into the nascent DNA. (F) Structure and color changes of cresol red under different pH. (G) Colorimetric detection of e13a2 fusion transcript by the naked eye and quantitative detection of e13a2 fusion transcript by three-primary colors (RGB) reading mode with an open-source App (Color Picker) on a SmartPhone. The RGB values of each reaction tubes can be obtained by adjusting the viewing position (black cross).

RESULTS AND DISCUSSION Overview of Vis-Fusion LIEXA method for fusion gene assay. The overview of Vis-Fusion LIEXA method for fusion gene detection is schematically illustrated in Figure 1. The BCR-ABL fusion gene is a well-known chimeric gene, encodes a hybrid BCR-ABL protein with increased tyrosine activity in CML.4, 27 The BCR gene has several breakpoints (Figure 1A), which can result in multiple fusion transcripts (Figure 1B). In this study, the BCR-ABL fusion transcript e13a2, containing exon 2-11 (a2-a11) from ABL gene and exon 1-13 (e1-e13) from the BCR gene, is employed as a model target. Firstly, two stemloop probes (SLP-e13 and SLP-a2) have been ingeniously designed. Each probe contains a stem-loop structure and a 20 nucleotides anti-target-specific sequence (I and II) respectively complementary to exon 13 of BCR and exon 2 of ABL at the fusion junction (Figure 1C). Only in the presence of e13a2, the SLP-e13 and SLP-a2 adjacently hybridized to the e13a2 target transcript at the fusion junction, the two probes can be ligated together with catalysis of SplintR ligase to form a double stemloop DNA, a distinctive DNA molecule, which can initiate the quickly and efficiently isothermal exponential amplification (IEXA) by using Bst WarmStart 2.0 DNA polymerase under isothermal conditions.28 The amplification processes are the most similar to the recycling amplification steps of loopmediated isothermal amplification (LAMP)29(Figure S1). On the other hand, in the presence of other fusion transcripts (such

as e1a2 e14a2 and e19a2), the SLP-e13 and SLP-a2 cannot be ligated and no subsequent amplification occurs due to the lacking of splint-template, however, these transcripts can also be amplified during PCR amplification by using a primer pair separately complementary to exon 13 of BCR gene and exon 2 of ABL gene, which greatly challenges to the specificity of PCR-based assay. The ingenious design can address all limitations of the PCR-based assay. The superior specificity of ligase guarantees the high specificity to effectively avoid interference from other fusion transcripts. And the rapid and efficient amplification feature of the LAMP provides ultrahigh sensitivity to quantify low-abundance fusion gene in a complex sample. In addition, anti-target-specific sequences (I and II) in the two stem-loop DNA probes can be flexibly designed according to interested other targets and the stem-loop sequences of the two probes can be designed identically, making different targets can be analyzed by using a universal primer pair, which greatly simplifies design of probes/primers and effectively reduces amplification bias arising from different target sequence. During the rapid and efficient amplification, by-products including pyrophosphates (PPi) and a hydrogen ion (H+) that are released as each dNTP is incorporated into the nascent DNA (Figure 1E). Under weakly buffered conditions, the H+ released by the efficient amplification will be enough to cause the drop in pH resulting color change of the pH-sensitive indicator, such as cresol red (Figure 1F), which has an easily discernable color



transition from red to yellow in the neutral range. Thus, the fusion gene transcript can be visually detected with the naked eye. More importantly, accurate quantification results can be obtained by three-primary colors (RGB) reading mode with an open-source App (Color Picker) on a SmartPhone (Figure 1G).

Figure 2. Feasibility evaluation of the Vis-Fusion LIEXA-based fusion assay. (A). Non-denaturing polyacrylamide gel electrophoresis (PAGE) analysis of IXEA reaction products (reaction time of IEXA reaction: 30 min). Lane 1 and lane 2: reaction products produced by 100 fM e13a2 and blank, respectively, under buffered condition. Lane 3 and lane 4: reaction products produced by 100 fM e13a2 and blank, respectively, under low buffered condition. Lane L: Double-strand (ds) DNA ladder. (B). pH values of the reaction system before initiation and after completion. (C). Colorimetric detection of fusion gene by using the Vis-Fusion LIEXA method. The blank was treated with the same procedures but without any transcripts.

Evaluation of the feasibility of the proposed Vis-Fusion LIEXA. Generally, there is no noticeable pH change during DNA polymerization reaction procedure due to the buffered conditions (10 mM-50 mM Tris-HCl), which provide the preferred conditions for DNA polymerase and neutralize the released H+ during dNTP incorporated into the nascent DNA. In this paper, to make released H+ causing pH change, we respectively prepared a low buffered (low concentration TrisHCl from ligase storage solution, about 100 µM in final ligation reaction) Ligation SUPER Mix for ligation reaction and a low buffered (low concentration Tris-HCl from Bst WarmStart 2.0 DNA polymerase storage solution, about 50 µM in final ligation reaction) WarmStart Colorimetric IEXA SUPER Mix for amplification reaction. Firstly, verification experiments were conducted to evaluate the feasibility of the Vis-Fusion LIEXA fusion gene assay. As displayed in Figure 2A, 100 fM e13a2 fusion transcript (lane 1 and lane 3) can produce multiple ladder-like amplification products of LAMP29 under buffered and low buffered conditions. In contrast, no such ladder-like products can be observed in the blank (lane 2 and lane 4). These results clearly verify that the ligation and amplification reactions can be efficiently carried out under low buffered conditions. To further support the feasibility of colorimetric detection for fusion gene by using the Vis-Fusion LIEXA system, the reaction pH before initiation and after completion was measured by precision pH-indicator strips. And an obvious drop in pH was observed from the initial alkaline pH (approximately 8.0-8.5) to a final acidic pH (about 6.5-7.0) (Figure 2B). This pH change is sufficient to cause the color change of cresol red with a pH transition range 8.8-7.2. As shown in Figure 2C, 100 fM e13a2 fusion transcript produced

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an obvious color change after performing 30 min IEXA reaction while the blank reaction maintained the initial color. All of these results strongly indicate that the Vis-Fusion LIEXA method is unquestionably feasible for visual detection of fusion gene.

Figure 3. (A) Color changes to different concentrations of e13a2 fusion transcript and blank. From left to right: 100 pM, 10 pM, 1 pM, 100 fM, 10 fM, 1 fM, 100 aM, and blank (0). (B) Calibration plots of -lgCe13a2(M) VS rate values of G/(R+G+B). The reaction time of IEXA reaction: 16 min. (C). The real-time fluorescence curves produced e13a2 fusion transcript with different concentration. From left to right, the e13a2 fusion transcript are successively 100 pM, 10 pM, 1 pM, 100 fM, 10 fM, 1 fM, 100 aM and 0 (Blank). (D). Calibration plots of -lgCe13a2(M) VS POI values of real-time fluorescence curve. Error bars represent the standard deviation of three replicates.

Analytical performance of the Vis-Fusion LIEXA for the detection of BCR-ABL fusion gene transcript. The VisFusion LIEXA method for fusion gene assay is based on the target-splinted ligation-triggered isothermal exponential amplification. So, the optimization of conditions of ligation and IEXA reaction have been investigated (Figure S2-S4). Under the optimized conditions, the dynamic range and sensitivity of the Vis-Fusion LIEXA-based for fusion transcript assay are evaluated to detect 10-fold serial dilutions of a synthetic e13a2 fusion transcript fragment as a target. As exhibited in Figure 3A, with the increasing of the e13a2 fusion transcript concentration, the color changes after 16 min IEXA reaction become more and more obvious indicating that the proposed method can be used for colorimetric detection of the fusion gene by the naked eye. In the classic RGB (0-255, 0-255, 0-255) color model, all colors perceived by the naked eye can be formed on screen by mixing the amounts of the R, G, and B color. So the color of each reaction can be represented by RGB values. In the proposed fusion gene assay, normalized RGB values (G/(R+G+B)) was used to represent color information of each reaction tubes, which can effectively eliminate RGB errors caused by different photographing conditions.30 More importantly, we first found that the G/(R+G+B) values are linearly dependent on the logarithm (lg) of the target concentration in the ranges 100 pM-100 fM and 100 fM-100 aM (Figure 3B). The correlation equations are G/(R+G+B)=0.508+0.0166lgCe13a2(M) (correlation coefficient R2=0.994) and G/(R+G+B)=0.722+0.0331lgCe13a2(M) (correlation coefficient R2=0.997), respectively. Furthermore,


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Table 1. Determination

of BCR-ABL fusion transcript e13a2 in total RNA from K562 cells

Sample (n=3)

Average amount e13a2 (zmol)

10 ng total RNA

213

10 ng total RNA + 800 zmol synthesized e13a2 transcript fragment

957

1 ng total RNA

20

1 ng total RNA + 80 zmol synthesized e13a2 transcript fragment

97

Figure 4. Specificity of the Vis-Fusion LIEXA-based method for detecting e13a2 transcript VS other BCR-ABL-related fusion transcripts (including e1a2, e14a2, and e19a2) by e13a2-specific DNA probes (SLP-e13 and SLP-a2). (A) Colors of different transcripts at different IXEA reaction times. (B) Non-denaturing polyacrylamide gel electrophoresis analysis of IXEA reaction products (reaction time of IEXA reaction: 30 min). Each concentration of transcript was 100 fM. The blank was treated with the same procedures but without any transcripts.

the RGB values can be easily and rapidly obtained by an opensource App with SmartPhone-assisted RGB reading mode. Meanwhile, under low buffered conditions, the synthetic e13a2 fusion transcript fragment was detected by a real-time fluorescence PCR instrument with SYBR Green I (SG) as the fluorescent intercalator.28 It can be seen that the fluorescence curves arise more rapidly with increasing concentrations of the target (Figure 3C) and the POI value (defined as the time corresponding to the maximum slope of real-time fluoresce curve) has a linear relationship with the lg of the target concentration in the ranges 100 pM-100 aM (Figure 3D). The fitted curve equation is POI=-14.2-2.09lgCe13a2(M) (correlation coefficient: R2=0.995). The performance for quantitative fusion gene of the SmartPhone-assisted manner is comparable to that of real-time manner. These results demonstrating that the VisFusion LIEXA is a reliable method for accurate quantitative analysis fusion gene at a wide range without expensive instruments. Specificity evaluation of the Vis-Fusion LIEXA. Generally, one gene may have several breakpoints, which can generate multiple fusion gene transcripts, such as the BCR-ABL fusion gene transcripts e1a2, e14a2, and e19a2, which have the same sequence (exon 2-11) from the ABL gene. (Figure 1B). It challenges the specificity of PCR-based fusion gene assay. So e1a2, e14a2, and e19a2 transcripts were selected as a challenging set to evaluate the specificity of the Vis-Fusion LIEXA method. As presented in Figure 4A, after performing 16 min and 30 min IEXA reaction, the desired color change appeared in e13a2 positive reaction tubes but all of the other transcripts tubes remained the starting color same as the color of the blank (without any transcript). Meanwhile, the IEXA

Recovery (%) 93.0 96.2

reaction products were analyzed by non-denaturing polyacrylamide gel electrophoresis (PAGE). As shown in Figure 4B, only in the presence of e13a2 fusion transcript, the IEXA reaction occurs efficiently accompanied with multiple ladder-like amplification products. When the presence of other transcripts, as same as blank, no such ladder-like products can be observed, which further confirms that the Vis-Fusion LIEXA assay is utterly dependent on specific ligation of the two stemloop probes at target fusion junction site. These results strongly suggested that the Vis-Fusion LIEXA assay exhibited high specificity for target fusion transcript detection even if other fusion transcripts coexist. Quantitative detection BCR-ABL fusion gene in total RNA samples. Finally, to assess the potential utility of the VisFusion LIEXA, the proposed method has been employed to detect BCR-ABL fusion transcript e13a2 in total RNA samples extracted from breast cancer cell line (MCF-7), normal cell human embryonic lung fibroblast (MRC-5), chronic myeloid leukemia cell line (K562). As shown in Figure 5, only the total RNA sample of K562 cell has apparent color changes and other samples, as well as the blank, have no color changes. The results suggest that BCRABL fusion transcripts e13a2 can be used as a molecular biomarker for CML. For this reason, the BCR-ABL fusion gene is used as gold standard molecular biomarkers by NCCN Guidelines® for CML diagnosis, therapy, and prognosis. According to the calibration curve (Figure 3B), the average amount of e13a2 transcript in 10 ng and 1 ng total RNA sample of K562 are 213 zmol and 20 zmol, respectively. Furthermore, recovery experiments were performed to evaluate the reliability of the proposed method for fusion gene assay, as shown in Table 1, the determined e13a2 transcript in the spiked samples was 957 zmol and 97 zmol with a recovery of 93.0% and 96.2%, respectively. These results clearly demonstrated that the Vis-Fusion LIEXA is a practical and reliable method for accurate quantitative detection of fusion gene transcript in a complex biological sample at a zmol level.

Figure 5. Colorimetric detection of the BCR-ABL fusion gene in total RNA extracted from different cell lines by using Vis-Fusion LIEXA method. The reaction time of IEXA reaction: 16 min.

CONCLUSIONS In summary, we have demonstrated that the proposed Vis-Fusion LIEXA method can be applied to the colorimetric detection of fusion gene by naked eye or accurate quantization fusion gene by SmartPhone-assisted RGB reading mode without other expensive instruments. By mean of this POCT method, as low as 2 zmol fusion gene can be rapidly visualized and quantified. In addition, the proposed assay also exhibits a great dynamic range over 6 orders of magnitude and high specificity to effectively avoid



interference from other homologous fusion transcripts. Furthermore, the Vis-Fusion LIEXA is a versatile method for visual and quantitative any DNA/RNA targets because the antitarget-specific sequences (I and II) in stem-loop DNA probes can be flexibly designed according to interested target. Particularly, the ligation reaction possesses superior specificity to efficiently distinguish the single-nucleotide difference in DNA/RNA targets,31-34 making the method also useful for mutation analysis. These features highlight the ability of the Vis-Fusion LIEXA and provide a perfect POCT platform for rapid, robust and sensitive detection of various nucleic acid biomarkers in clinical molecular diagnosis, even in home diagnosis.

ASSOCIATED CONTENT Supporting Information The sequences of DNA and RNA oligonucleotides (Table S1), the recycling amplification steps of LAMP (Figure S1), and optimization of the experimental conditions (Figure S2-S4) are presented in the Supporting Information. The Supporting Information is available free of charge on the ACS Publications website:

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21705008 and 21775012), China Postdoctoral Science Foundation (2017M620605), Fundamental Research Funds for the Central Universities (FRF-TP-18-011A1).

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Vis-Fusion LIEXA: A Point-of-Care Testing Method for Highly Specific

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