Primer combination and method for detecting EGFRT790M mutation in free DNA and application

Abstract

The invention belongs to the technical field of genes, and particularly relates to a primer combination and method for detecting EGFR T790M mutation in free DNA and application. The primer combination is used for specifically amplifying EGFR T790M in free DNA, and comprises the following four primers: a forward primer, a reverse primer, a forward primer, a reverse primer and a reverse primer, wherein the forward primer is nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2; a reverse primer: a nucleotide sequence as shown in SEQ ID NO: 3; and a retardant primer: a nucleotide sequence as shown in SEQ ID NO: 4. The detection method comprises the following steps: (1) ultralow-frequency mutation PCR (Polymerase Chain Reaction) amplification: mixing the primer group with a DNA template to be detected, and carrying out multiple PCR amplification cycles; and (2) carrying out electrophoresis on the amplification product, and carrying out concentration determination on the recovered gel after electrophoresis. Compared with the prior art, the detection sensitivity can be improved, the operation process is simplified, and the detection cost is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of gene technology, and particularly relates to a primer combination, method and application for detecting EGFR T790M mutation in cell-free DNA. Background Art

[0002] EGFR is a transmembrane receptor that participates in various biological processes such as cell proliferation, differentiation, and survival. In many cancers, mutations or overexpression of EGFR are closely related to the occurrence and development of tumors. Among the mechanisms of acquired resistance to EGFR-TKI, the EGFR gene T790M mutation accounts for approximately 50%.

[0003] The T790M mutation is located in exon 20 of the EGFR gene, specifically, the threonine (T) at amino acid position 790 is replaced by methionine (M). This mutation is generally considered an acquired mutation that occurs after a patient receives EGFR-targeted therapy (such as erlotinib, gefitinib), leading to drug resistance in tumor cells. The T790M mutation is a common resistance mechanism in EGFR-mutation-positive patients after receiving first-line EGFR inhibitor therapy, and approximately 50-60% of resistance cases are related to this mutation. Detecting the T790M mutation is very important for guiding subsequent treatment, especially when a patient experiences disease progression. It can be detected by tissue biopsy or liquid biopsy (blood sample). Targeted drugs for the T790M mutation, such as osimertinib, have been approved for the treatment of patients with EGFR T790M-mutation-positive non-small cell lung cancer. Research on the EGFR T790M mutation continues, and scientists are exploring new targeted therapies and combination treatment strategies to overcome drug resistance and improve treatment efficacy. The EGFR T790M mutation is an important resistance mechanism in non-small cell lung cancer, and understanding its characteristics and clinical significance is crucial for optimizing the treatment plan for patients.

[0004] With the rapid development of gene detection technology, the detection methods for the T790M mutation site of the EGFR gene have been constantly updated. Currently, the common detection methods mainly include: Amplified refractory mutation system (ARMS), Super-amplified refractory mutation system (Super-ARMS), Next-generation sequencing (NGS), and Droplet digital PCR (ddPCR). ARMS PCR is also known as allele-specific PCR, with a sensitivity of about 1%, simple operation and low cost, and it is the standard method for hospital tissue biopsy. However, for samples with a mutation abundance less than 1%, the sensitivity of ARMS PCR is very low, and there is a possibility of false-negative detection. The Super-ARMS PCR technology uses a hairpin primer instead of the linear primer in ordinary ARMS PCR, changes its thermodynamic properties, improves the ability to identify single nucleotide mutations, reduces non-specific amplification, and the sensitivity is increased from 1% to 0.2%. Next-generation sequencing technology or high-throughput sequencing technology (NGS) is famous for allowing parallel sequencing of millions to billions of DNA molecules at one time, with a detection limit of 0.1%. However, the NGS experimental process is complex, the detection cycle is long, and professional bioinformatics personnel are required to analyze a large amount of data. Therefore, it is generally not the first choice for single gene mutation detection in clinical practice. ddPCR dilutes the PCR reaction finitely, distributes a sample into hundreds to tens of thousands of different reaction units, amplifies in different reaction chambers, and then statistically analyzes and quantifies according to the Poisson distribution principle. It is an absolute quantification technology for nucleic acid molecules. Some research reports show that ddPCR can detect a mutation frequency as low as 0.01% on the premise that there is enough starting template amount in the sample and the experimental design is appropriate. It is currently the detection technology with the highest sensitivity in detecting the T790M mutation, but the instruments and reagents used are expensive and not universal. Therefore, there is an urgent need to find a method for detecting the EGFR T790M mutation with high specificity, simple process, low cost, and high sensitivity.

[0005] Therefore, there is still a need in the art to seek a method for detecting the EGFR T790M mutation to improve the diagnostic accuracy rate, simplify the operation process, increase the sensitivity, and reduce the detection cost. Summary of the Invention

[0006] (I) Technical problems to be solved

[0007] The object of the present invention is to provide a primer combination, method and application for detecting EGFRT790M mutation in cell-free DNA to solve at least one of the above problems, so as to solve the problems of high detection limit, low sensitivity and high cost in detecting EGFR T790M mutation in cell-free DNA in the prior art, and achieve the effects of improving the diagnostic accuracy rate, simplifying the operation process, increasing the sensitivity and reducing the detection cost.

[0008] (II) Technical Solution

[0009] The object of the present invention is achieved by the following technical solutions:

[0010] The first aspect of the present invention discloses a primer combination for detecting EGFR T790M mutation in cell-free DNA. The primer group is used for specifically amplifying EGFR T790M in cell-free DNA and comprises the following 4 primers:

[0011] Forward primer: nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2;

[0012] Reverse primer: nucleotide sequence shown in SEQ ID NO:3;

[0013] Blocker primer: nucleotide sequence shown in SEQ ID NO:4.

[0014] Furthermore, the blocker primer is completely complementary to the wild-type EGFR template.

[0015] Furthermore, the blocker primer preferentially binds to the wild-type template during the PCR amplification process.

[0016] Furthermore, the blocker primer can inhibit the amplification efficiency of the wild-type template, while allowing the forward primers (SEQ ID NO:1 and SEQ ID NO:2) to bind to the mutant template, and differentially enrich the T790M mutant through competitive amplification.

[0017] The second aspect of the present invention discloses a method for detecting EGFR T790M mutation in cell-free DNA by using the above-mentioned primer combination. The detection method is characterized in that the detection method comprises the following steps:

[0018] (1) Ultra-low frequency mutation PCR amplification: Mix the primer group with the DNA template to be detected and perform multiple rounds of PCR amplification cycles;

[0019] (2) Take the amplification product for electrophoresis, and measure the concentration after recovering the gel after electrophoresis.

[0020] Further, during the multi-round PCR cycling process, the wild-type amplification efficiency is reduced and the amplification of the mutant template is enhanced by the preferential binding of the blocker primer to the wild-type template; through the cumulative difference in the amplification efficiency of multiple PCR cycles, exponential enrichment of the mutant DNA with the EGFR T790M mutation is achieved.

[0021] Further, the PCR amplification process is as follows: pre-denaturation at 98°C for 4 min, followed by 30 - 50 cycles, each cycle consisting of denaturation at 98°C for 20 s, annealing at 60°C for 30 s, extension at 72°C for 150 s, and finally thorough extension at 72°C for 5 min, and stored at 4°C.

[0022] Further, the number of cycles is preferably 50.

[0023] Further, the molar concentration ratio of the forward primers SEQ ID NO:1 and SEQ ID NO:2 to the blocker primer SEQ ID NO:4 is 1:(5 - 15), preferably 1:10.

[0024] Further, the concentration is measured by a spectrophotometer and then Sanger sequencing is performed.

[0025] Further, the lower limit of detection of the EGFR T790M mutation obtained by the detection method reaches a variant allele frequency (VAF) of 0.03%.

[0026] The third aspect of the present invention discloses an application for detecting the EGFR T790M mutation in cell-free DNA as described above, and the application includes detecting the EGFR T790M mutation in cell-free DNA in blood, cerebrospinal fluid or tissue fluid and being used in combination with a digital PCR (ddPCR) or next-generation sequencing (NGS) platform.

[0027] Further, the mutation detection can be used to monitor the drug resistance of patients with non-small cell lung cancer, colorectal cancer or glioblastoma.

[0028] (III) Beneficial effects

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] (1) The present invention inhibits the amplification efficiency of the wild-type DNA template by designing a blocker primer, while allowing the forward primer to bind to the mutant template. During the PCR amplification process, the blocker primer preferentially binds to the wild-type template, reducing its amplification efficiency, while the mutant template is enriched through competitive amplification. After multiple rounds of PCR cycles, this difference in amplification efficiency gradually accumulates, and eventually achieves exponential enrichment of EGFR T790M mutant DNA. Traditional PCR methods can usually only detect mutation frequencies of 1%-5%, while the present invention increases the detection sensitivity to 0.03% VAF, significantly improving the accuracy of detection.

[0031] (2) The present invention involves PCR amplification by adding forward primers, reverse primers and blocker primers. The blocker primer preferentially binds to wild-type DNA, while the specific primer accurately targets the mutation region. This dual mechanism ensures that the amplified product is derived only from the target mutation, avoiding interference from non-target sequences, thereby improving the reliability and specificity of the test results.

[0032] (3) The primer combination of the present invention is suitable for conventional PCR amplification, and does not require complex equipment or special instruments. By simply adding a special primer combination, efficient enrichment of mutant DNA and improved detection sensitivity can be achieved. The method has a simple operation process, low cost, and can be directly used in kit development, providing an efficient and economical solution for the clinical detection of EGFR T790M mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 The electrophoresis quality inspection diagram of the product of the reference sample amplified with 0.1% VAF by the primer combination of Example 1 of the present invention and Comparative Examples 1-3; Figure 1 (a) From left to right are Marker (BBI, B500331), amplification product of Example 1, amplification product of Comparative Example 1, amplification product of Comparative Example 2, and amplification product of the wild-type control of Comparative Example 3; Figure 1 (b) is a comparison chart of the bp lengths corresponding to each marker band.

[0035] Figure 2 The Sanger sequencing results of the products of the reference sample with 0.1% VAF amplified by the primer combination of Example 1 of the present invention and Comparative Examples 1-3; wherein Figure 2 (a) is the Sanger sequencing result of the amplified product of Example 1;Figure 2 (b) is the Sanger sequencing result of the amplification product of Comparative Example 1; Figure 2 (c) is the Sanger sequencing result of the amplification product of Comparative Example 2; Figure 2 (d) is the Sanger sequencing result of the amplification product of Comparative Example 3.

[0036] Figure 3 This is the product electrophoresis quality inspection chart of the EGFR T790M reference samples with different VAFs amplified in Example 1 of the present invention; from left to right are Marker (BBI, B500331), 0.1% VAF amplification product, Marker (BBI, B500331), 0.05% VAF amplification product, 0.03% VAF amplification product, 0.01% VAF amplification product, and each VAF sample was replicated 3 times technically.

[0037] Figure 4 This is the Sanger sequencing result of the products of the EGFR T790M reference samples with different VAFs amplified in Example 1 of the present invention; among them Figure 4 (a) is the Sanger sequencing result of the 0.1% VAF product amplified in Example 1; Figure 4 (b) is the Sanger sequencing result of the 0.05% VAF product amplified in Example 1; Figure 4 (c) is the Sanger sequencing result of the 0.03% VAF product amplified in Example 1; Figure 4 (d) is the Sanger sequencing result of the 0.01% VAF product amplified in Example 1. Detailed implementation manners

[0038] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] The following specific examples illustrate the implementation manners of the present application. Those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, without conflict, the following embodiments and the features in the embodiments can be combined with each other. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts belong to the scope of protection of the present application.

[0040] Note that the following description pertains to various aspects of embodiments within the scope of the appended claims. It should be apparent that the aspects described herein can be embodied in a wide variety of forms, and any specific structure and / or function described herein is merely illustrative. Based on this application, those skilled in the art should understand that one aspect described herein can be implemented independently of any other aspect, and two or more of these aspects can be combined in various ways. For example, any number and aspects set forth herein can be used to implement a device and / or practice a method. Additionally, this device can be implemented and this method can be practiced using other structures and / or functionality in addition to one or more of the aspects set forth herein.

[0041] In addition, in the following description, specific details are provided to facilitate a thorough understanding of the examples. However, those skilled in the art will understand that the examples can be practiced without these specific details.

[0042] Unless otherwise specified, the raw materials or devices used in the following examples are commercially available raw materials or conventional experimental devices.

[0043] The present invention provides a primer combination and method for detecting the EGFR T790M mutation in cell-free DNA. The primer set is used for specifically amplifying EGFR T790M in cell-free DNA and comprises the following 4 primers:

[0044] Forward primer: nucleotide sequences as shown in SEQ ID NO:1 and SEQ ID NO:2;

[0045] Reverse primer: nucleotide sequence as shown in SEQ ID NO:3;

[0046] Blocker primer: nucleotide sequence as shown in SEQ ID NO:4.

[0047] Among them, the blocker primer is completely complementary to the wild-type EGFR template and preferentially binds to the wild-type template during the PCR amplification process, which can inhibit the amplification efficiency of the wild-type template, while allowing the forward primers (SEQ ID NO:1 and SEQ ID NO:2) to bind to the mutant template, and differentially enrich the T790M mutant through competitive amplification.

[0048] The method for detecting the EGFR T790M mutation in cell-free DNA comprises the following steps:

[0049] (1) Ultra-low frequency mutation PCR amplification: Mix the primer set with the DNA template to be tested and perform multiple rounds of PCR amplification cycles;

[0050] (2) Take the amplification product for electrophoresis, and after electrophoresis, recover the gel for concentration determination.

[0051] The following describes the technical solutions provided by each embodiment of the present application in conjunction with the accompanying drawings.

[0052] (1) Primer combination control experiment

[0053] Example 1

[0054] In this Example 1, the mutation detection of EGFR T790M was performed on a reference sample with 0.1% VAF. The specific process is as follows:

[0055] 1. Use TaKaRa Taq TM Hot Start Version (Takara, R007Q) to amplify EGFR T790M. The primer combination and sequences are shown in Table 1, the reaction system is shown in Table 2, and the reaction program is shown in Table 3;

[0056] Table 1 EGFR T790M PCR amplification primers in Example 1

[0057]

[0058] Table 2 Amplification system

[0059]

[0060] Table 3 Amplification program

[0061]

[0062] 2. After the amplification is completed, take 5 μL of the amplification product, add 1 μL of 6× Loading Buffer, mix well and perform 2.0% agarose gel electrophoresis to observe the amplification result. The result is as Figure 1 shown; for the gel recovery of the PCR product, use the Axygen AxyPrep DNA Gel Extraction Kit (AP-GX-500) and perform the gel recovery operation according to the kit instructions.

[0063] 3. Measure the concentration of the recovered product using a ultra-micro spectrophotometer, and then perform Sanger sequencing. The sequencing result is shown in Figure 2 (a).

[0064] Comparative Example 1

[0065] In this Comparative Example 1, the mutation detection of EGFR T790M was performed on a reference sample with 0.1% VAF. The specific process is as follows:

[0066] 1. Use TaKaRa Taq TMThe Hot Start Version (Takara, R007Q) was used to perform PCR amplification on EGFR T790M. The primer combination and sequences are shown in Table 4. Compared with Example 1, the forward primer shown in SEQ ID NO:2 was not added to the primers in this comparative example. The reaction system and reaction program of Comparative Example 1 were the same as those of Example 1;

[0067] Table 4 Primers for EGFR T790M PCR Amplification in Comparative Example 1

[0068]

[0069] 2. After the amplification was completed, 5 μL of the amplification product was taken, 1 μL of 6× Loading Buffer was added, and after mixing, 2.0% agarose gel electrophoresis was performed to observe the amplification result. The result is as Figure 1 shown; The PCR product was recovered using the Axygen AxyPrep DNA Gel Extraction Kit (AP-GX-500) according to the kit instructions for gel extraction operation.

[0070] 3. The concentration of the recovered product was measured using a ultra-micro spectrophotometer, and then Sanger sequencing was performed. The sequencing result is shown in Figure 2 (b).

[0071] Comparative Example 2

[0072] In this Comparative Example 2, the mutation detection of EGFR T790M was performed on a reference sample with 0.1% VAF. The specific process is as follows:

[0073] 1. TaKaRa Taq TM Hot Start Version (Takara, R007Q) was used to perform PCR amplification on EGFR T790M. The primer combination and sequences are shown in Table 5. Compared with Example 1, the forward primer shown in SEQ ID NO:1 was not added to the primers in this comparative example. The reaction system and reaction program of Comparative Example 2 were the same as those of Example 1;

[0074] Table 5 Primers for EGFR T790M PCR Amplification in Comparative Example 2

[0075]

[0076]

[0077] 2. After the amplification was completed, 5 μL of the amplification product was taken, 1 μL of 6× Loading Buffer was added, and after mixing, 2.0% agarose gel electrophoresis was performed to observe the amplification result. The result is as Figure 1As shown in the figure; for the gel extraction of PCR products, the AxyPrep DNA Gel Extraction Kit (AP-GX-500) from Axygen was used, and the gel extraction operation was carried out according to the kit instructions.

[0078] 3. The concentration of the recovered product was measured using a ultra-micro spectrophotometer, and then Sanger sequencing was performed. The sequencing results are shown in Figure 2 (c).

[0079] Comparative Example 3

[0080] In this Comparative Example 3, the mutation detection of EGFR T790M was carried out on a reference sample with 0.1% VAF. The specific process is as follows:

[0081] 1. TaKaRa Taq TM Hot Start Version (Takara, R007Q) was used for PCR amplification of EGFR T790M. The primer combinations and sequences are shown in Table 6. Compared with Example 1, the blocker primer shown in SEQ ID NO: 4 was not added to the primers in this comparative example. The reaction system and reaction program of Comparative Example 3 were the same as those of Example 1;

[0082] Table 6 Primers for EGFR T790M PCR Amplification in Comparative Example 3

[0083]

[0084] 2. After the amplification was completed, 5 μL of the amplification product was taken, 1 μL of 6× Loading Buffer was added, and after mixing, 2.0% agarose gel electrophoresis was carried out to observe the amplification results. The results are as Figure 1 shown; for the gel extraction of PCR products, the AxyPrep DNA Gel Extraction Kit (AP-GX-500) from Axygen was used, and the gel extraction operation was carried out according to the kit instructions.

[0085] 3. The concentration of the recovered product was measured using a ultra-micro spectrophotometer, and then Sanger sequencing was performed. The sequencing results are shown in Figure 2 (d).

[0086] According to Figure 1 and Figure 2 in the results, primer combination 1 can amplify the reference sample with 0.1% VAF and has a good inhibition effect, and basically no wild-type peak pattern appears. Primer combination 2 can amplify the reference sample with 0.1% VAF, but cannot completely inhibit the wild type, showing a heterozygous peak pattern with coexistence of mutant and wild sites. Primer combination 3 cannot amplify the reference sample with 0.1% VAF. Primer combination 4 can amplify the wild-type sample, and the sequencing results show that the target mutation was not detected.

[0087] (2) Mutation detection of different VAF samples

[0088] The primer combinations described in Example 1 were used to amplify VAF samples with different ratios (VAF samples of 0.1%, 0.05%, 0.03% and 0.01%) respectively for mutation detection of EGFR T790M. TaKaRa Taq TM Hot Start Version (Takara, R007Q) was used for PCR amplification of EGFR T790M. The primer combinations and sequences are shown in Table 1, the reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0089] After amplification, 5 μL of the amplification product was taken, 1 μL of 6× Loading Buffer was added, and after mixing, 2.0% agarose gel electrophoresis was carried out to observe the amplification result. The result is as Figure 3 shown; The PCR product was recovered from the gel using the Axygen AxyPrep DNA Gel Extraction Kit (AP-GX-500), and the gel extraction operation was carried out according to the kit instructions.

[0090] The concentration of the recovered product was measured using an ultra-micro spectrophotometer, and then Sanger sequencing was carried out. The sequencing result is shown in Figure 4 .

[0091] From Figure 3 , Figure 4 it can be seen that by detecting the amplicons of 0.1%, 0.05%, 0.03% and 0.01% VAF samples, it was found that the target mutation could be detected in samples within the range of 0.1% - 0.01% VAF. Among them, both wild type and mutant existed in the 0.01% VAF sample, indicating that the amplification of the wild type in the 0.01% VAF sample was not completely inhibited, but the amplification of the wild type in the 0.03% VAF sample was completely inhibited. Therefore, the detection limit of the EGFR T790M mutation detected in the present invention reached 0.03% VAF.

[0092] For the same or similar parts among the various embodiments in this specification, reference can be made to each other. Each embodiment focuses on the differences from other embodiments.

[0093] The above description is only the specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed in the present application should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A primer combination for detecting EGFR T790M mutation in free DNA, characterized in that: The primer combination is used to specifically amplify EGFR T790M in free DNA, and comprises the following 4 primers: Forward primer: nucleotide sequence as shown in SEQ ID NO: 1 and SEQ ID NO: 2; Reverse primer: the nucleotide sequence shown in SEQ ID NO: 3; Blocker primer: the nucleotide sequence shown in SEQ ID NO:

4.

2. A primer combination for detecting EGFR T790M mutation in free DNA according to claim 1, characterized in that: The blocker primer is fully complementary to the wild-type EGFR template.

3. A primer combination for detecting EGFR T790M mutation in free DNA according to claim 2, characterized in that: The blocker primer preferentially binds to the wild-type template during PCR amplification.

4. A method for detecting EGFR T790M mutation in free DNA using the primer combination as claimed in claim 1, characterized in that: The detection method comprises the following steps: (1) Ultra-low frequency mutation PCR amplification: the primer set is mixed with the DNA template to be tested and multiple rounds of PCR amplification cycles are performed; (2) The amplified product is subjected to electrophoresis and the gel is recovered for concentration determination.

5. The method for detecting EGFR T790M mutation in free DNA according to claim 4, characterized in that: During the multiple rounds of PCR cycles, the blocker primer preferentially binds to the wild-type template, thereby reducing the wild-type amplification efficiency and enhancing the amplification of the mutant template; and the exponential enrichment of the mutant DNA of the EGFR T790M mutation is achieved through the cumulative difference in amplification efficiency of multiple PCR cycles.

6. The method for detecting EGFR T790M mutation in free DNA according to claim 4, characterized in that: The PCR amplification process is as follows: pre-denaturation at 98°C for 4 minutes, followed by 30-50 cycles, each cycle comprising denaturation at 98°C for 20 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 150 seconds, and finally complete extension at 72°C for 5 minutes, and then stored at 4°C.

7. The method for detecting EGFR T790M mutation in free DNA according to claim 4, characterized in that: The molar concentration ratio of the forward primers SEQ ID NO: 1 and SEQ ID NO: 2 to the blocker primer SEQ ID NO: 4 is 1:(5-15).

8. The method for detecting EGFR T790M mutation in free DNA according to claim 4, characterized in that: Concentration determination was performed by spectrophotometry followed by Sanger sequencing.

9. A use of detecting EGFR T790M mutation in free DNA as claimed in claim 1, characterized in that: The application includes detecting the EGFR T790M mutation in free DNA in blood, cerebrospinal fluid or interstitial fluid.

10. The use of detecting EGFR T790M mutation in free DNA according to claim 9, characterized in that: The mutation assay can be used to monitor drug resistance in patients with non-small cell lung cancer, colorectal cancer or glioblastoma.

Images