One-pot EB virus nucleic acid detection method and device based on RPA-PfAgo

Abstract

The invention discloses a one-pot EB virus nucleic acid detection method and device based on RPA-PfAgo, and belongs to the technical field of biological detection. The method comprises the following steps: physically isolating an RPA amplification system from a PfAgo cleavage reaction system in the same container, adding a sample to be detected, carrying out RPA amplification at 37-42 DEG C, heating to 85-96 DEG C, and releasing physical isolation to carry out PfAgo cleavage reaction. According to the method, isothermal amplification (RPA), specific nucleic acid recognition based on PfAgo and fluorescence readout are integrated in a single closed reaction tube, the reaction tube does not need to be opened, the pollution risk is remarkably reduced, rapid, sensitive and visual EBV detection is achieved, and the method is suitable for field detection, rapid virus detection in a resource limited environment and nucleic acid detection application of other similar pathogens.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method based on RPA- Pf Ago's one-pot EB virus nucleic acid detection method and device. Background Technology

[0002] Epstein-Barr virus ( Epstein–Barr virus Human herpesvirus 4 (HHV-4), also known as EBV, was first discovered in Burkitt lymphoma in 1964 and is the first human carcinogenic virus to be identified.

[0003] EBV is associated with various malignant tumors and autoimmune diseases. While current diagnostic methods offer high analytical sensitivity, they require complex nucleic acid extraction procedures, expensive equipment, and highly skilled personnel, limiting their application in point-of-care testing (POCT). Molecular detection technologies, such as polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR), are widely used for clinical pathogen detection (including EBV) due to their high sensitivity and quantitative capabilities. However, these methods rely on sophisticated thermal cycling equipment, rigorous nucleic acid extraction procedures, and highly trained operators, resulting in long testing cycles and high equipment costs, making them unsuitable for resource-constrained or rapid on-site testing. Isothermal amplification techniques (such as LAMP and RPA) can rapidly amplify nucleic acids under isothermal conditions, offering simple operation and making them suitable as the core amplification strategy for point-of-care testing. However, isothermal amplification (especially RPA) is prone to producing non-specific amplification products or false positives, requiring subsequent specific identification to improve accuracy. CRISPR / Cas systems (such as Cas12 / Cas13) have been used in conjunction with isothermal amplification to construct highly specific detection platforms. However, CRISPR typically relies on RNA-type guide sequences (crRNA), which are expensive to prepare and less stable than DNA at room temperature. Furthermore, some Cas enzymes require recognition of PAM sequences, which limits target selection and increases design complexity and cost.

[0004] Pyrococcus furiosus Argonaute ( Pf Ago is a programmable endonuclease that guides DNA through 5′-phosphorylation to precisely cleave single-stranded DNA. Unlike CRISPR nucleases, Pf Ago does not require a neighboring motif as a guide, and its guide molecule is DNA rather than RNA, thus offering greater flexibility and lower cost. Recent studies have demonstrated that, based on... Pf Ago's diagnostic testing methods possess high specificity, even achieving single-base resolution; however, publicly available... Pf Ago detection methods all employ a "amplification-capping transfer-detection" workflow, because... Pf Ago at higher Mn2+ The activity is optimal under certain concentration conditions, while high concentrations of Mn... 2+ Typically, RPA enzyme activity is inhibited, necessitating separate reaction systems to accommodate the differences in conditions. This two-step process increases operational complexity, prolongs detection time, and significantly raises the risk of inter-tube contamination and false positives. Summary of the Invention

[0005] 1. Purpose of the invention To address the aforementioned problems, the purpose of this invention is to provide a method based on RPA- Pf Ago's one-pot EB virus nucleic acid detection method and device combines isothermal amplification and... Pf Ago testing is performed in the same closed reaction system, thus achieving one-pot, closed, rapid, and low-contamination EBV nucleic acid testing.

[0006] 2. Technical Solution To achieve the objective of this invention, the technical solution adopted by this invention is as follows: This invention provides an RPA- Pf Ago's one-pot EB virus nucleic acid detection method combines the RPA amplification system and... Pf The Ago cleavage reaction system was physically isolated. After adding the sample to be tested, RPA amplification was first performed between 37-42℃, and then heated to 85-96℃ to break the physical isolation. Pf Ago cleavage reaction.

[0007] Furthermore, the RPA amplification system described above contains a set of specific primers.

[0008] Furthermore, the above Pf The Ago cleavage reaction system contains Mn 2+ , Pf Ago enzyme, 2 gDNA strips, fluorescent probe.

[0009] Furthermore, the specific primer sequences described above are shown in SEQ ID NO.1 and SEQ ID NO.6.

[0010] Furthermore, the above gDNA sequences are shown in SEQ ID NO.16 and SEQ ID NO.17.

[0011] Furthermore, the above fluorescent probe sequence is shown in SEQ ID NO.20.

[0012] Furthermore, the above Pf Ago cleaves Mn in the reaction system 2+ The concentration is 0.25 mM.

[0013] Furthermore, the above PfThe concentration of gDNA in the Ago cleavage reaction system is 0.5 µM.

[0014] Furthermore, the above Pf The dosage of Ago enzyme is 400U.

[0015] Furthermore, the RPA amplification time mentioned above is 25 minutes.

[0016] Furthermore, the volume of the above RPA amplification system is 10 µL.

[0017] Furthermore, the above Pf The Ago cutting reaction time is 30 minutes. This invention provides a method based on RPA- Pf The overall process of Ago's one-pot EB virus nucleic acid testing method is as follows: Figure 1 As shown. The detection process includes four consecutive steps: sample pretreatment, RPA-based amplification, ... Pf Ago-mediated cleavage and fluorescence signal reading. The entire detection process is performed in a single sealed tube, where RPA amplification and... Pf The Ago cleavage reaction is initially separated by a layer of paraffin. During the RPA stage, the target EBV sequence is amplified exponentially; subsequently, heating to 95°C melts the paraffin, causing it to rise and allowing the amplified product to interact with the surrounding material. Pf The Ago cleavage reaction mixture was thoroughly mixed. Guided by 5′ phosphorylated gDNA, Pf Ago specifically cleaves the RPA amplification product between nucleotides 10 and 11 of its complementary sequence. These cleavage products then act as new gDNA, triggering... Pf Ago performs secondary cleavage of the fluorescent group-quenching probe, thereby releasing the FAM fluorescent signal. Finally, the fluorescence signal can be monitored in real time by a fluorescence detector, or observed directly under blue or ultraviolet light.

[0018] The present invention also provides a nucleic acid detection device, which uses specific materials in a container to combine the RPA amplification system and... Pf Ago cleaves the reaction system physically isolated, Pf The Ago cleavage reaction system is located beneath a specific material and contains Mn. 2+ , Pf Ago enzyme, gDNA, and fluorescent probe; the RPA amplification system is located above a specific material and contains a set of specific primer sequences; the specific material is solid below 50°C and liquid or gaseous above 70°C.

[0019] Furthermore, the container described above is a transparent plastic tube.

[0020] Furthermore, the container described above is a PCR tube.

[0021] Furthermore, the density of the aforementioned specific material is less than that of water.

[0022] Furthermore, the aforementioned specific material is paraffin wax.

[0023] 3. Beneficial effects Compared with the prior art, the advantages of this invention are as follows: (1) This invention provides a method based on RPA- Pf Ago's one-pot EB virus nucleic acid detection method and device, which combines isothermal amplification (RPA) and based on... Pf Ago integrates specific nucleic acid recognition and fluorescence readout into a single sealed reaction tube, making it suitable for on-site testing, rapid virus detection in resource-constrained environments, and nucleic acid detection applications for other similar pathogens.

[0024] (2) This invention provides a method based on RPA- Pf Ago's one-pot EB virus nucleic acid detection method is simple to operate, requires low equipment (only a constant temperature heating device is needed), has a short detection time (≤1 hour), high sensitivity (near qPCR level), and is visual (observed with the naked eye or a simple light source).

[0025] (3) This invention provides a method based on RPA- Pf Ago's one-pot EB virus nucleic acid detection method and device achieves isothermal amplification and... Pf Ago-mediated specific cleavage / fluorescence readout eliminates the need to open the reaction tube, significantly reducing the risk of contamination and enabling rapid, sensitive, and visualized EBV detection.

[0026] (4) This invention provides a method based on RPA- Pf Ago's one-pot EB virus nucleic acid detection method can be extended to detect other viruses, bacteria, or specific mutation sites by changing the target sequence and primers / gDNA.

[0027] (5) This invention provides a method based on RPA- Pf Ago's one-pot EB virus nucleic acid detection method and device supports real-time monitoring with fluorescence instruments or direct visual observation of endpoint results under LED blue light / ultraviolet light, making it suitable for use in resource-constrained scenarios. Attached Figure Description

[0028] Figure 1 This is a diagram illustrating the principle and flowchart of a single-tube detection system.

[0029] Figure 2 This is a sequence diagram of the conserved region of EBV.

[0030] Figure 3 This is a schematic diagram of recombinant plasmid construction.

[0031] Figure 4 This is a graph showing the screening results of RPA primers, gDNA, and probe pairs.

[0032] Figure 5 This is a graph showing the results of optimized reaction conditions.

[0033] Figure 6 This is a graph showing the sensitivity and specificity results.

[0034] Figure 7 This is a diagram of the clinical sample validation results. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0037] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0038] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0039] As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0040] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0041] Sample source statement: Peripheral blood samples were collected from patients in the Oncology and Hematology departments of the Third Affiliated Hospital of Soochow University and stored at -20°C. The study protocol was approved by the Institutional Review Board of the Third Affiliated Hospital of Soochow University, and informed consent was waived. All procedures were performed in accordance with the principles of the Declaration of Helsinki.

[0042] In this invention, statistical analysis was performed using GraphPad Prism 8.0 software with two-tailed t-tests. All experiments were repeated three times, and results are expressed as mean ± standard deviation (SD).

[0043] Construction of recombinant plasmids: This invention selects the BamHI-W region, which is highly conserved in the EBV genome and exists in different viral strains, as the amplification target. Figure 2 The sequence is shown in SEQ ID NO.22. Based on this, we specifically synthesized a fragment of the BamHI-W region and cloned it into the pUC57 vector, successfully constructing the recombinant plasmid pUC57-EBV (…). Figure 3 The formula for calculating plasmid copy number is as follows: copies / µL = (6.02 × 10 23 ) × (ng / µL × 10 -9 ) / (length of DNA × 660).

[0044] Subsequently, the plasmid concentration gradient was adjusted to 10 by successive 10-fold dilutions. 4 Up to 10 0 Copy / microliter, to meet the needs of subsequent experimental research.

[0045] The sequences involved in this invention are shown in Table 1 below: Table 1

[0046] All oligonucleotides in the table were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The 12 gDNAs were phosphorylated using T4 polynucleotide kinase (New England Biolabs, Ipswich, MA).

[0047] Example 1 This embodiment provides a one-pot RPA- Pf Preparation of the Ago detection kit.

[0048] S1: Add to PCR tube PfAgo cleavage reaction mixture (20 µL) contains: 2.25 µL 100 mM MnCl2, 3 µL Pf Ago enzyme (Beijing Baoying Tonghui Biotechnology Co., Ltd., JY0316), 3 µL 10 µM phosphorylated gDNA, 3 µL 10 µM FAM / BHQ1 labeled probe, 3 µL 10× buffer, and 5.75 µL ddH2O.

[0049] S2: Cover 10 µL of molten paraffin (melting point 60°C) with a solution heated to 95°C. Pf Apply the Ago mixture to the surface of the liquid, ensuring the paraffin layer covers the entire surface. Once the test tube has cooled to room temperature, the paraffin layer will solidify.

[0050] S3: Add 9 µL of RPA amplification mixture onto the solidified paraffin layer. The RPA amplification system was prepared as follows: using a DNA amplification kit (Leshan Biotechnology (Wuxi) Co., Ltd.). First, redissolve the RPA powder in 25 µL of buffer, then add 2 µL of forward primer (10 µM), 2 µL of reverse primer (10 µM), and 13.5 µL of ddH2O; next, take 8.5 µL of this mixture and add it onto the paraffin layer, followed by 0.5 µL of 280 mM MgOAc buffer. Seal the PCR tube and store it at -20°C.

[0051] Example 2 This embodiment provides the one-pot RPA method from Embodiment 1. Pf Instructions for using the Ago test kit.

[0052] Genomic DNA was extracted using a commercial whole blood DNA extraction kit (Suzhou Kuangyuan Biomolecular Technology Co., Ltd.). The blood sample and lysis reagent were thoroughly mixed at a volume ratio of 1:4 (v / v) and then incubated at 95 °C for 5 min to achieve cell / virus lysis and release of nucleic acids.

[0053] Remove the detection PCR tube prepared in Example 1 and thaw it overnight at 4°C. Add 1 µL of DNA template and begin the incubation program with two temperature gradients: The reaction was first carried out at 37 °C for 25 minutes to complete RPA amplification, followed by a reaction at 95 °C for 30 minutes. The assay was performed using a SLAN-96S real-time PCR system for easy recording of fluorescence intensity. Throughout the process, the fluorescence signal was recorded every minute, and the reaction endpoint was observed by LED blue light illumination. A positive result was considered achieved if the fluorescence intensity of the sample was higher than that of the template-free control (NTC).

[0054] Example 3 This embodiment provides screening of RPA primers.

[0055] RPA primers are shown in Table 1. RPA amplification was performed using a DNA amplification kit (Leshan Biotechnology (Wuxi) Co., Ltd.) in a 50µL reaction system. The specific components were: 25µL 2× rehydration buffer, 2µL each of 10µM forward and reverse primers (10µM), 5µL DNA template, 2.5µL 280mM magnesium acetate, and an appropriate amount of nuclease-free water to bring the total volume to the required level. After mixing, the mixture was briefly vortexed and centrifuged, then incubated at 37 ℃ for 30 minutes. The amplified products were subjected to 2% agarose gel electrophoresis at 120V for 30 minutes, and the results were observed under UV light.

[0056] Experimental results are as follows Figure 4 As shown in A, the amplified fragment size ranged from 100 to 250 bp. The F1 / R3 primer pair produced the strongest amplification product (152 bp) with no nonspecific bands, and was therefore selected for subsequent experiments.

[0057] Example 4 This embodiment provides the design and screening of gDNA and probes.

[0058] Based on the RPA amplification products obtained in Example 3, 12 guide DNAs (gDNA, 16 nucleotides each) and 2 fluorescent probes (5′-FAM / 3′-BHQ1) were designed, as shown in Table 1.

[0059] Pf The Ago cleavage reaction system (20µL) includes: 2µL 10× buffer, 2µL Pf Ago enzyme (Beijing Baoying Tonghui Biotechnology Co., Ltd.), 0.25mM MnCl2, 2µM RPA amplification product, 2µM gDNA, and 1µM probe were used. The reaction was incubated at 95℃ for 30 minutes in a SLAN-96S real-time PCR system (Shanghai Hongshi Medical Technology Co., Ltd.).

[0060] Six combinations of gDNA and probes were designed for testing:

[0061] like Figure 4 B, the highest fluorescence signal produced by different gDNA combinations (represented by vertical bars in the bar chart). The error bars represent the standard error of three parallel replicates. All combinations effectively mediated... Pf Ago cleaves single-stranded DNA templates (RPA products denatured to ssDNA by heating to 95°C); however, the combination of gDNA9 and gDNA10 with probe 2 exhibited the highest cleavage efficiency. This combination was selected for use in the optimized RPA- Pf Ago testing system.

[0062] Example 5 This embodiment provides a one-pot RPA- Pf Parameter optimization of the Ago detection method.

[0063] The reaction parameters were systematically optimized, including: Pf Ago enzyme dosage (0-2000U), below the paraffin layer Pf The concentration of gDNA in the Ago reaction system (0-2µM), below the paraffin layer Pf Mn in the Ago reaction system 2+ Concentration (0-5mM), RPA reaction time (5-40 minutes), RPA amplification volume (10-30µL), and Pf Ago reaction time (10-70 minutes).

[0064] The experimental procedures were the same as in Examples 1 and 2 (excluding the -20℃ storage and thawing steps). The F1 / R3 primer pair, gDNA9, and gDNA10 were selected to be used with probe 2. During optimization, only one parameter was changed at a time, while the remaining parameters remained constant. The fluorescence signal was continuously monitored at 95℃ for 30 minutes.

[0065] Mn 2+ The concentration adjustment results showed that the strongest fluorescence signal was produced at 0.25 mM. Figure 5 A).

[0066] The gDNA concentration was found to be 0.5 µM, at which the maximum cleavage activity was obtained. Figure 5 B).

[0067] Enzyme dosage: 400U Pf Ago generates the highest signal, but excessive enzyme actually reduces cleavage efficiency. Figure 5 C).

[0068] RPA amplification showed a detectable signal after 15 minutes and reached a plateau at 25 minutes. Extending the incubation time did not provide additional gain, therefore 25 minutes was determined to be the optimal amplification time. Figure 5 D).

[0069] RPA amplification volume: Signals produced in the range of 15–30 µL are similar, while the 10 µL system, although with slightly weaker fluorescence, has been identified as the most cost-effective choice without affecting detection reliability. Figure 5 E).

[0070] PfThe Ago cleavage reaction time results showed that the fluorescence intensity increased rapidly in the first 20 minutes, stabilized at 30 minutes, and then showed no further enhancement. Figure 5 F).

[0071] In summary, the optimal one-pot RPA method... Pf The Ago cleavage reaction conditions are: Mn 2+ The concentration was 0.25 mM, and the gDNA concentration was 0.5 µM. Pf The Ago dosage was 400 U, the RPA amplification time was 25 minutes, and the RPA amplification volume was 10 µL. Pf The Ago cutting reaction time is 30 minutes.

[0072] Example 6 This embodiment describes a one-pot RPA process. Pf The sensitivity of Ago detection was tested.

[0073] The pUC57-EBV plasmid was diluted with ddH2O (the concentration range after dilution was 10). 0 Up to 10 4 The analytical sensitivity was evaluated using copies / µL, with ddH2O used as a negative control. Each concentration was tested in triplicate. Experimental procedures were performed according to Examples 1 and 2 (excluding the -20°C storage and thawing steps), and the detection parameters used the optimal reaction conditions obtained in Example 5.

[0074] The results showed that the limit of detection (LOD) for EBV in this system could reach 1 copy / μL ( Figure 6 A), and the fluorescence intensity at this concentration was statistically significantly different from that of the negative control.

[0075] Example 7 This embodiment describes a one-pot RPA process. Pf Ago tests are performed to detect specificity.

[0076] Collect EBV, cytomegalovirus (CMV), adenovirus ( Adenovirus Mycoplasma pneumoniae ( ) M. pneumoniae DNA was extracted from positive clinical samples for RPA testing and hepatitis B virus (HBV) to verify RPA- Pf The specificity of the Ago method was assessed. Nucleic acids were extracted using the TIANamp Genomic DNA Kit (Tiangen Biotech (Beijing) Co., Ltd.), with 5 µL of extract (50 ng / µL) used per reaction. Experimental procedures were performed according to Examples 1 and 2 (excluding the -20°C storage and thawing steps), and the optimal reaction conditions obtained in Example 5 were used for detection. After quantitative fluorescence quantification, images of each test tube were acquired under ultraviolet (UV) and blue light.

[0077] The results showed that only EBV templates exhibited significant fluorescence signals, while other pathogens showed no signals, confirming the good specificity of this method. Figure 6 B).

[0078] Example 8 This embodiment provides a one-pot RPA process. Pf Clinical validation of Ago testing.

[0079] The clinical validation used 31 peripheral blood samples from patients suspected of EBV infection. Each test was pre-prepared in 20µL. Pf The Ago cutting reaction mixture was coated with 10 µL of paraffin. After the paraffin solidified, 8.5 µL of RPA mixture, 1 µL of processed clinical DNA sample, and 0.5 µL of buffer B were added to bring the total reaction volume (excluding paraffin) to 30 µL. The entire reaction was incubated at 37°C for 25 minutes (RPA amplification), followed by heating at 95°C for 30 minutes (paraffin melting and promoting amplification). Pf (Ago digestion). The test tubes were kept sealed throughout the entire experiment.

[0080] As a control, we also used a commercially available EBV detection kit (Guangzhou DaAn Gene Co., Ltd., DA0153) and performed quantitative PCR (qPCR) detection according to the manufacturer's instructions. The qPCR program included: incubation at 50°C for 30 minutes, incubation at 93°C for 2 minutes, followed by 10 cycles (93°C for 45 seconds, 55°C for 60 seconds), and then 30 cycles (93°C for 30 seconds, 55°C for 45 seconds). EBV detection was performed via the FAM fluorescence channel.

[0081] RPA- Pf The detection results from the Ago platform are as follows: Figure 7 As shown in Figure A, 21 samples tested positive and 10 tested negative; the fluorescence detection results were consistent with the visible signals observed under ultraviolet and blue light. Figure 7 B). qPCR analysis results are as follows: Figure 7 C (21 positive, 10 negative). These results indicate that RPA- Pf The Ago assay is accurate, rapid, and cost-effective in EBV detection, and its performance is comparable to that of qPCR.

Claims

1. A method based on RPA- Pf Ago's one-pot EB virus nucleic acid detection method is characterized by... The RPA amplification system and Pf The Ago cleavage reaction system was physically isolated. After adding the sample to be tested, RPA amplification was first performed between 37-42℃, and then heated to 85-96℃ to break the physical isolation. Pf Ago cleavage reaction; the RPA amplification system contains a set of specific primers; The Pf The Ago cleavage reaction system contains Mn 2+ , Pf Ago enzyme, 2 gDNA strips, fluorescent probe.

2. The detection method according to claim 1, characterized in that, The specific primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.6; the gDNA sequences are shown in SEQ ID NO.16 and SEQ ID NO.17; and the fluorescent probe sequences are shown in SEQ ID NO.

20.

3. The detection method according to claim 1 or 2, characterized in that, The Pf Ago cleaves Mn in the reaction system 2+ The concentration was 0.25 mM, and the gDNA concentration was 0.5 µM. Pf The dosage of Ago enzyme is 400U.

4. The detection method according to claim 3, characterized in that, The RPA amplification time was 25 minutes and the RPA amplification volume was 10 µL.

5. The detection method according to claim 4, characterized in that, The Pf The Ago cutting reaction time is 30 minutes.

6. A nucleic acid detection device, characterized in that, The RPA amplification system and... are combined in a container using specific materials. Pf The Ago cleavage reaction system is physically isolated, the Pf The Ago cleavage reaction system is located beneath a specific material and contains Mn. 2+ , Pf Ago enzyme, gDNA, and fluorescent probe; the RPA amplification system is located above a specific material and contains a set of specific primer sequences; the specific material is solid below 50°C and liquid or gaseous above 70°C.

7. The nucleic acid detection device according to claim 6, characterized in that, The container is a transparent plastic tube.

8. The nucleic acid detection device according to claim 7, characterized in that, The container is a PCR tube.

9. The nucleic acid detection device according to claim 6, characterized in that, The specific material has a density less than that of water.

10. The nucleic acid detection device according to claim 9, characterized in that, The specific material is paraffin wax.

Images