Polymerase Chain Reaction and Dot-Blot Hybridization for Leptospira Detection in Water Samples

Summary

In this study, a dot-blot application was designed to detect Leptospira from the three main clades in water samples. This method allows for the identification of minimal DNA quantities specifically targeted by a digoxigenin-labeled probe, easily detected by an anti-digoxigenin antibody. This approach is a valuable and satisfactory tool for screening purposes.

Abstract

The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by probe hybridization in the presence of carrier DNA. It is based on the transfer of a known amount of DNA onto an inert solid support, such as a nylon membrane, utilizing the dot-blot apparatus and without electrophoretic separation. Nylon membranes have the advantage of high nucleic acid binding capacity (400 µg/cm²), high strength, and are positively or neutrally charged. The probe used is a highly specific ssDNA fragment of 18 to 20 bases long labeled with digoxigenin (DIG). The probe will conjugate with the Leptospira DNA. Once the probe has hybridized with the target DNA, it is detected by an anti-digoxigenin antibody, allowing its easy detection through its emissions revealed in an X-ray film. The dots with an emission will correspond to the DNA fragments of interest. This method employs the non-isotopic labeling of the probe, which may have a very long half-life. The drawback of this standard immuno-label is a lower sensitivity than isotopic probes. Nevertheless, it is mitigated by coupling polymerase chain reaction (PCR) and dot-blot assays. This approach enables the enrichment of the target sequence and its detection. Additionally, it may be used as a quantitative application when compared against a serial dilution of a well-known standard. A dot-blot application to detect Leptospira from the three main clades in water samples is presented here. This methodology can be applied to large amounts of water once they have been concentrated by centrifugation to provide evidence of the presence of Leptospiral DNA. This is a valuable and satisfactory tool for general screening purposes, and may be used for other non-culturable bacteria that may be present in water, enhancing the comprehension of the ecosystem.

Introduction

Leptospirosis in humans mainly originates from environmental sources^1,2. The presence of Leptospira in lakes, rivers, and streams is an indicator of leptospirosis transmission among wildlife, and domestic and production animals that may eventually come into contact with these bodies of water^1,3,4. Furthermore, Leptospira has been identified in non-natural sources, including sewage, stagnant and tap water^5,6.

Leptospira is a worldwide distributed bacteria^7,8, and the role of the environment in its preservation and transmission has been well recognized. Leptospira can survive in drinking water under variable pH and minerals⁹, and in natural bodies of water¹. It can also survive for long periods in distilled water¹⁰, and under constant pH (7.8), it may survive up to 152 days¹¹. Moreover, Leptospira may interact in bacterial consortiums to survive harsh conditions^12,13. It may be part of biofilms in freshwater with Azospirillum and Sphingomonas and is even capable of growing and enduring temperatures exceeding 49 °C^14,15. It can also multiply in waterlogged soil and remain viable for up to 379 days¹⁶, preserving its ability to cause the disease for as long as a year^17,18. However, little is known about the ecology within water bodies and how it is distributed within them.

Since its discovery, the study of the genus Leptospira was based on serological tests. It was not until the current century that molecular techniques became more prevalent in the study of this spirochaete. The dot-blot has been scarcely used for its identification using (1) an isotopic probe based on the 16S rRNA and on an inter-simple sequence repeat (ISSR)^19,20, (2) as a nanogold based immunoassay for human leptospirosis applied to urine²¹, or (3) as an antibody-based assay for bovine urine samples²². The technique fell in disuse because it was originally based on isotopic probes. However, it is a well-known technique that, coupled with PCR, yields enhanced results, and it is considered safe due to the use of non-isotopic probes. PCR plays a crucial role in the enrichment of the Leptospira DNA by amplifying a specific DNA fragment that may be found in trace amounts in a sample. During each PCR cycle, the amount of the targeted DNA fragment is doubled in the reaction. At the end of the reaction, the amplicon has been multiplied by a factor of more than a million²³. The product amplified by PCR, often not visible in agarose electrophoresis, becomes visible through specific hybridization with a DIG-labeled probe in the dot-blot^24,25,26.

The dot-blot technique is simple, robust, and suitable for numerous samples, making it accessible to laboratories with limited resources. It has been employed in a variety of bacteria studies, including (1) oral bacteria²⁷, (2) other sample types such as food and feces²⁸, and (3) the identification of unculturable bacteria²⁹, often in agreement with other molecular techniques. Among the advantages offered by the dot-blot technique are: (1) The membrane has a high binding capacity, capable of binding over 200 μg/cm² of nucleic acids and up to 400 μg/cm²; (2) Dot-blot results can be visually interpreted without requiring special equipment, and (3) they can be conveniently stored for years at room temperature (RT).

The genus Leptospira has been classified into pathogenic, intermediate, and saprophytic clades^30,31. The distinction among these clades can be achieved based on specific genes such as lipL41, lipL32, and the 16S rRNA. LipL32 is present in the pathogenic clades and exhibits high sensitivity in various serological and molecular tools, whereas it is absent in saprophyte species²¹. The housekeeping gene lipL41 is known for its stable expression and used in molecular techniques³², while the 16S rRNA gene is utilized for their classification.

This methodology can be applied to large volumes of water once they have been concentrated by centrifugation. It allows the assessment of various points and depths within a water body to detect the presence of leptospiral DNA and the clade to which it belongs. This tool is valuable for both ecological and general screening purposes and can also be employed to detect other non-culturable bacteria that may be present in water.

Additionally, PCR and dot-blot assays are technically and economically affordable to a wide range of laboratories, even those lacking sophisticated or expensive equipment. This study aims to apply the digoxigenin-based dot-blot for the identification of the three Leptospira clades in water samples collected from natural bodies of water.

Bacterial strains
Twelve Leptospira serovars (Autumnalis, Bataviae, Bratislava, Canicola, Celledoni, Grippothyphosa, Hardjoprajitno, Icterohaemorrhagiae, Pomona, Pyrogenes, Tarassovi, and Wolffi) were included in this study. These serovars are part of the collection at the Department of Microbiology and Immunology, Faculty of Veterinary Medicine and Zootechnics, National Autonomous University of Mexico, and they are currently used in the microagglutination test (MAT).

All Leptospira serovars were cultured in EMJH, and their DNA was extracted using a commercial DNA extraction kit (see Table of Materials). A genomic DNA mix of the twelve serovars was used as a positive control for the Leptospira pathogenic clade. As a positive control of the Leptospira intermediate clade, genomic DNA from Leptospira fainei serovar Hurstbridge strain BUT6 was included, and as a positive control for the Leptospira saprophyte clade, genomic DNA of Leptospira biflexa serovar Patoc strain Patoc I, was also included.

Negative controls consisted of an empty plasmid, DNA from non-related bacteria (Ureaplasma urealyticum, Staphylococcus aureus, Brucella abortus, Salmonella typhimurium, Shigella boydii, Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli), and PCR-grade water, which served as non-template control.

Water samples
Twelve trial grab samples were collected using a stratified-haphazard sampling method from the Cuemanco Biological and Aquaculture Research Center (CIBAC) (19° 16' 54" N 99° 6' 11" W). These samples were obtained at three depths: superficial, 10, and 30 cm (Figure 1A, B). The water collection procedures did not impact any endangered or protected species. Each sample was collected in a sterile 15 mL microcentrifuge tube. To collect the sample, each tube was gently submerged in the water, filled at the selected depth, and then sealed. The samples were maintained at 22 °C and promptly transported to the laboratory for processing.

Each sample was concentrated by centrifugation in sterile 1.5 mL microcentrifuge tubes at 8000 x g for 20 min at room temperature. This step was repeated until all the samples were concentrated into one tube, which was then used for DNA extraction (Figure 1C).

Figure 1: Concentration of water samples by centrifugation. (A) Water sampling ponds, and (B) Natural streams. (C) Centrifugation-based water sample processing in repeated steps as many times as needed (n). Please click here to view a larger version of this figure.

DNA extraction
Total DNA was isolated using a commercial Genomic DNA kit according to the manufacturer's instructions (see Table of Materials). DNA extractions were eluted in 20 µL of elution buffer, and DNA concentration was determined by a UV Spectrophotometer at 260-280 nm, and stored at 4 °C until use.

PCR amplification
The PCR targets were the 16S rRNA, lipL41, and lipL32 genes, which identify DNA from the genus Leptospira and allow the distinction among the three clades: pathogenic, saprophytic, and intermediate. Both primers and probe designs were based on the previous works by Ahmed et al., Azali et al., Bourhy et al., Weiss et al., and Branger et al.^{33,34,35,36,37}. The sequence of each probe, primer, and amplified fragment are described in Table 1, and their alignment with reference sequences are provided in Supplementary File 1, Supplementary File 2, Supplementary File 3, Supplementary File 4, and Supplementary File 5. The PCR reagents and thermocycling conditions are described in the protocol section.

Amplification products were visualized by electrophoretic separation on a 1% agarose gel in TAE (40 mM Tris base, 20 mM Acetic acid, and 1 mM EDTA; pH 8.3), at 60 V for 45 min with ethidium-bromide detection, as shown in Supplementary Figure 1. Genomic DNA obtained from each serovar was used with concentrations ranging from 6 x 10⁶ to 1 x 10⁴ genomic equivalent copies (GEq) in each PCR reaction, based on the genome size of L. interrogans (4, 691, 184 bp)³⁸ for pathogenic Leptospira, the genome size of L. biflexa (3, 956, 088 bp)³⁹ for saprophytic Leptospira, and the genome size of L. fainei serovar Hurstbridge strain BUT6 (4, 267, 324 bp) with accession number AKWZ00000000.2.

The sensitivity of the probes was assessed with DNA from each pathogenic serovar, L. biflexa serovar Patoc strain Patoc I, and L. fainei serovar Hurstbridge strain BUT6 in each experiment. To assess the specificity of the PCR and dot-blot hybridization assay, DNA from non-related bacteria was included.

Table 1: PCR primers and probes to amplify products for identifying the pathogenic, saprophyte, and intermediate clades of Leptospira. Please click here to download this Table.

Dot-blot hybridization assay
The technique is called dot-blot because the holes in which the DNA sample is placed have a dot shape, and when they are sucked to be fixed in place by vacuum suction, they acquire this shape. This technique was developed by Kafatos et al.⁴⁰. The technique allows the semi-quantification of Leptospira in each PCR-positive sample. The protocol consists of a denaturation with NaOH 0.4 M at room temperature, samples with Leptospira DNA from 30 ng to 0.05 ng, corresponding to 6 x 10⁶ to 1 x 10⁴ leptospires, are blotted onto a nylon membrane with a 96-well dot-blot apparatus. After immobilization, the DNA is bound to the membrane by exposure to 120 mJ UV light. Each DNA probe is conjugated with digoxigenin-11 dUTP by a terminal transferase catalysis step at the 3' end (Digoxigenin is a plant steroid obtained from Digitalis purpurea, used as a reporter⁴¹). Following the stringent hybridization of the labeled DNA probe (50 pmol) at the specific temperature onto the target DNA, the DNA hybrids are visualized by the chemiluminescence reaction with the anti-digoxigenin alkaline phosphatase antibody covalently conjugated with its substrate CSPD. The luminescence is captured by exposure to an X-ray film (Figure 2).

Figure 2: Steps of the procedure for the PCR-dot-blot assay. Please click here to view a larger version of this figure.

Protocol

1. Sample preparation

1. Concentrate each water sample in 1.5 mL microcentrifuge tubes by centrifugation at 8,000 x g for 10 min at 4 °C. Repeat this step, as many times as required, to concentrate the sample into a volume of 250 µL.
2. Use the DNA extraction kit according to the manufacturer´s instructions (see Table of Materials).
3. Perform the specific PCR according to the dot-blot probe that will be used (Table 1).
   1. Perform the amplifications in PCR tubes with the final volume of 25 µL containing 1 X buffer, 2.5 units of Taq polymerase, 1 µM of each primer, 0.2 mM of each dNTP, 1.5 mM MgCl₂, and 100 ng of target DNA from each water sample or reference genomic-DNA.
   2. Program the PCR reaction in a thermocycler according to the thermocycling conditions (Table 2).
   3. Store the reactions at 4 °C until use.
4. Place 10 µL of each PCR product to be blotted in a separate well of a 96-well plate.
5. Add 40 µL of TE to each well and mix by pipetting.
   NOTE: The 96-well plate can be sealed and stored at 4 °C overnight. Follow the instructions provided in Supplementary File 6 to prepare buffers and solutions for the protocol. Figure 3 depicts the steps of sample preparation.

Table 2: PCR thermocycling conditions for the 16S, lipL41, and lipL32 genes. Please click here to download this Table.

Figure 3: PCR and sample preparation. Applying the specific PCR protocol, the PCR product was transferred to a microtiter plate, and 40 µL of TE was added to each well. Please click here to view a larger version of this figure.

2. Assembling the dot-blot apparatus

NOTE: The assembling of the dot-blot apparatus is shown in Figure 4. During the procedure, wear gloves to handle the alkali solutions and protect the nylon membrane from contamination.

Figure 4: Dot-blot apparatus assembling. The filter paper and the nylon membrane (previously moistened in 10 X SSPE) must be arranged in the correct order. The assembly must be secured with the screws tightly before applying the vacuum. Each well needs to be washed with TE, and the PCR products are loaded into their respective wells. After transferring the PCR product through the membrane, each well is washed again with TE and allowed to dry. Please click here to view a larger version of this figure.

1. Cut the nylon membrane (see Table of Materials) and filter paper into sheets of size 12 x 8.5 cm.
2. Mark the membrane with a permanent marker.
3. Make a notch with scissors at one edge to indicate the right orientation. Use the mark to remember the sample order.
4. Moisten the membrane and filter paper with 10 X Saline-sodium phosphate-EDTA buffer (SSPE; 3 M NaCl, 0.2 M NaH₂PO₄, and 0.02 M EDTA, pH 7.4) (Supplementary File 6). Handle the membrane with a clean, blunt-ended tweezer.
5. Assemble the dot-blot chamber; first, the filter paper, followed by the membrane over the plastic seal. Secure the cover with the screws in a crosswise manner.
6. Connect the chamber to the vacuum, place 100 µL of TE in each well, hold the vacuum for 1 min, and then stop it.
7. Switch on the vacuum at low speed and load 50 µL of each sample into the corresponding well on the membrane of the dot-blot apparatus (following the pre-decided membrane distribution).
   NOTE: Homogenize each sample in the hemagglutination 96 well plate prior to placing it in the dot-blot chamber.
8. Allow the vacuum to dry the membrane. If necessary, hit the chamber gently to release the bubbles in the sample.
9. Wash each well by placing 100 µL of TE into it with a continuous vacuum and allowing it to dry.
   NOTE: After completing the transfer, it is crucial to first switch off the pump and detach it. Failure to do so may result in reflux entering the apparatus. DNA does not need a denaturation step if a positively charged nylon membrane is used, but if using other inert support, a pre-denaturation step and alkali-based denaturation may be needed⁴¹.

3. DNA denaturation and fixation

NOTE: Figure 5 illustrates the DNA membrane fixation procedure.

Figure 5: DNA-membrane fixation procedure. The DNA is denatured in an alkaline solution. Next, it is neutralized with 10 X SSPE, and the membrane is dried. Next, the membrane is transilluminated. The membrane is rehydrated with 2 X SSPE and pre-hybridized overnight. Please click here to view a larger version of this figure.

1. Incubate the membrane with 0.4 M NaOH for 10 min at room temperature.
2. Equilibrate the membrane with 10 X SSPE for 10 min at room temperature.
3. Dry the membrane at 80 °C for 2 h.
4. Transilluminate with 120 mJ UV light for 3 min. Ensure that the samples are oriented face down towards the UV light source. If using a UV-crosslinker, confirm that the samples are facing upward and repeat this step twice.
   NOTE: The membrane must be completely dry before UV crosslinking. DNA will be immobilized by a covalent bond to the nylon membrane. Each cross-linking takes about 18'' to 1' with an optimal UV irradiation dose, for most hybridization experiments, of approximately 0.6-0.8 kJ/m².
   Exposure to UV irradiation is harmful to the eyes and skin. Wear appropriate protective equipment and avoid exposure to bare skin.
5. Wash the membrane with 2 X SSPE for 10 min.
6. Carefully fold the membrane and insert it into the hybridization tube (use a 15 mL microcentrifuge tube).
7. Add 10 mL of the pre-hybridization solution (Supplementary File 6) to the tube and incubate at 42 °C overnight. Ensure that the tube has been properly sealed to prevent any leakage.

4. Hybridization

1. Remove 5 mL of the pre-hybridization solution from the hybridization tube. To use the 5 mL for a second time, keep it in another tube and store it at -4 °C.
2. Add 15 µL of the labeled probe (Table 1) to the hybridization tube. Rinse the tip into the solution to ensure that the labeled probe was thoroughly instilled into the dilution.
   NOTE: The DNA probe's binding is not as strong as the antigen-antibody binding. Therefore, changes in the concentration of the probe or DNA in the sample can influence the intensity of the emitted fluorescence. DNA probes are stoichiometric, meaning that the number of probe molecules bound to DNA is equivalent to the number of DNA molecules present in the solution. Figure 6 depicts the probe hybridization.
3. Incubate at 42 °C overnight.
4. Perform DNA probe DIG labeling. Follow the instructions specified in Table 3 to mix the reagents, then incubate the mixture at 37 °C for 1 h. Next, add 80 µL of distilled water and store the tailed probe at 4 °C.

Figure 6: Probe hybridization. The volume of the hybridization buffer is adjusted, and the digoxigenin-labeled probe is incorporated to allow the probe's hybridization overnight. Please click here to view a larger version of this figure.

Table 3: Reagents for the probes labeling with digoxigenin (DIG). Please click here to download this Table.

5. Chemiluminescence (anti-DIG tagging)

1. Wash the membrane twice with 2 X SSPE/0.1% SDS at room temperature for 10 min.
2. Incubate the membrane with 5 X SSPE/0.1% SDS at the probe's annealing temperature for 15 min.
   NOTE: In this step, use a container with a lid to maintain the temperature as long as possible. Ensure uniform and constant temperature across the membrane. The approximate volume in each wash is 100 mL of the solution indicated.
3. Wash the membrane once with Buffer 1 (Supplementary File 6) at room temperature for 5 min.
4. Wash the membrane once with Buffer 2 (Supplementary File 6) at room temperature for 30 min.
5. Wash the membrane with Buffer 2 and add the anti-digoxigenin antibody (15 µL) (see Table of Materials), then incubate for 45 min (the incubation time may vary from 30-60 min). This secondary antibody tags the probe for detection.
   NOTE: The membrane may be stored in Buffer 2 with the anti-digoxigenin antibody overnight at 4-8 °C. Anti-DIG tagging of the chemiluminescence process is depicted in Figure 7.

Figure 7: Anti-DIG tagging of the chemiluminescence process. The unbonded nucleic acids are removed with buffer solutions. The probe is aligned with the target DNA, and the excess is removed. The membrane is blocked with the blocking 1 X buffer, and the anti-DIG antibody is added (1:10000). Please click here to view a larger version of this figure.

6. Chemiluminescence (substrate application)

1. Wash the membrane twice in Buffer 1 at room temperature for 15 min.
2. Wash once in Buffer 3 (see Supplementary file 6) at room temperature for 5 min, allowing the membrane to drain most of the buffer, leaving it almost dry.
3. Add CSPD ready to use (Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro) tricyclo [3.3.1.13,7] decan}-4-yl) phenyl phosphate, see Table of Materials), and let stay for 5 min, (with damped fingers homogenize the CSPD from one side to the other).
4. Place the membrane in a transparent plastic bag (12.5 x 9 cm) that fits the membrane dimension. Carefully remove air bubbles, evenly distribute the CSPD, and heat-seal the bag.
5. Incubate the membrane in a water bath at 37 °C for 15 min (it can be up to 30 min), and ensure that the membrane with samples is placed downwards so that it is well submerged. Ensure that the temperature remains constant and well distributed over the membrane.
6. Dry the plastic bag and fix it with scotch tape on an already-used radiographic film. This will allow easy handling during the exposure procedure.
   NOTE: Figure 8 shows the substrate application of the chemiluminescence process.

Figure 8: Substrate application of the chemiluminescence process. The free antibody is removed, and the substrate CSPD (1:250) is added to the membrane. The reaction is activated by incubation at 37 °C and the membrane is arranged to record the chemiluminescence in an X-ray film. Please click here to view a larger version of this figure.

7. Chemiluminescence (detection)

1. Perform the exposure procedure. In the darkroom, prepare the developer and fixer solutions (see Table of Materials) in the respective trays.
2. In the dark, carefully place the fixed membrane facing a new radiographic film and insert it into a radiographic cassette (see Table of Materials).
3. Register the time of exposure. The time of exposure may vary from 1 min to 30 min or more. Start with 5 min and adjust the time accordingly.
4. Damp the X-ray film in developer solution for 1-3 min and rinse gently with tap water (15-45 s).
5. Damp the X-ray film in fixer solution for 1-3 min and rinse gently with tap water (45 s).
6. Allow the X-ray film to air dry and document the assay in a visible white lightbox.
   NOTE: Avoid shaking the membrane during the X-ray exposure time. The detection process is depicted in Figure 9.
7. Proceed to the result interpretation. In the exposed X-ray film, the dots with emission can be visually located and correspond to the DNA fragments with the probe's hybridization. The intensity of the emitted signal depends on the luminescence and the duration of the exposure.
   NOTE: Membranes can be stored dried between filter paper for several months at room temperature.

Figure 9: Detection of the chemiluminescence process. Under dark conditions, the membrane is exposed to an X-ray film inside an X-ray cassette. Next, it was allowed to stand during the exposition time, and then the X-ray film was developed and fixed. Finally, it was air dried and interpreted. Please click here to view a larger version of this figure.

8. Membrane de-hybridization procedure

1. Wash the membrane twice for 10 min with distilled water.
2. Wash the membrane for 20 min with 0.4 M NaOH at 53 °C (twice).
3. Wash twice the membrane for 10 min with 2 X SSPE.
4. Allow the membrane to dry at room temperature.
5. Keep the membrane and return to step 3.5 of the protocol.
   NOTE: The membrane may be stored in the pre-hybridization buffer overnight at 4-8 °C. The next day, restart with an incubation at 42 °C for 1 h, allowing the pre-hybridization buffer to reach the temperature. Then, proceed to step 3.5 of the protocol.

Results

To assess the effectiveness of the technique, genomic DNA from pure cultures of each Leptospira serovar was used, along with the clade-specific probe. Membranes were prepared with 100 ng of genomic DNA per PCR reaction for each serovar, followed by eight genomic DNA of non-related bacteria and variable concentrations of genomic DNA of the ad hoc Leptospira serovars. Each assay included positive, negative, and non-template control. These non-related genomic DNA did not show an affinity for the dot-blot p...

Discussion

The critical steps of the dot-blot technique include (1) DNA immobilization, (2) blocking of the free binding sites on the membrane with non-homologous DNA, (3) the complementarity between the probe and the target fragment under annealing conditions, (4) removal of the unhybridized probe, and (5) the detection of the reporter molecule⁴¹.

The PCR-Dot-blot has certain limitations, such as the technique does not provide information about the size of the hybridized fragment...

Materials

Name	Company	Catalog Number	Comments
REAGENTS
Purelink DNA extraction kit	Invitrogen	K182002
Gotaq Flexi DNA Polimerase (End-Point PCR Taq polymerase kit)	Promega	M3001
Whatman filter paper, grade 1,	Merk	WHA1001325
Nylon Membranes, positively charged Roll 30cm x 3 m	Roche	11417240001
Anti-Digoxigenin-AP, Fab fragments Sheep Polyclonal Primary-antibody	Roche	11093274910
Medium Base EMJH	Difco	S1368JAA
Leptospira Enrichment EMJH	Difco	BD 279510
Blocking Reagent	Roche	11096176001
CSPD ready to use Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro) tricyclo [3.3.1.13,7] decan}8-4-yl) phenyl phosphate	Merk	11755633001
Deoxyribonucleic acid from herring sperm	Sigma Aldrich	D3159
Developer Carestream	Carestream Health Inc	GBX5158621
Digoxigenin-11-ddUTP	Roche	11363905910
EDTA, Disodium Salt (Dihydrate)	Promega	H5032
Ficoll 400	Sigma Aldrich	F8016
Fixer Carestream	Carestream Health Inc	GBX 5158605
Lauryl sulfate Sodium Salt (Sodium dodecyl sulfate; SDS) C12H2504SNa	Sigma Aldrich	L5750
N- Lauroylsarcosine sodium salt CH3(CH2)10CON(CH3) CH2COONa	Sigma Aldrich	L-9150	It is an anionic surfactant
Polivinylpyrrolidone (PVP-40)	Sigma Aldrich	PVP40
Polyethylene glycol Sorbitan monolaurate (Tween 20)	Sigma Aldrich	9005-64-5
Sodium Chloride (NaCl)	Sigma Aldrich	7647-14-5
Sodium dodecyl sulfate (SDS)	Sigma Aldrich	151-21-3
Sodium hydroxide (NaOH)	Sigma Aldrich	1310-73-2
Sodium phosphate dibasic (NaH2PO4)	Sigma-Aldrich	7558-79-4
Terminal transferase, recombinant	Roche	3289869103
Tris hydrochloride (Tris HCl)	Sigma-Aldrich	1185-53-1
SSPE 20X	Sigma-Aldrich	S2015-1L	It can be Home-made following Supplementary File 6
Primers	Sigma-Aldrich	On demand	Follow table 1
Probes	Sigma-Aldrich	On demand	Follow table 1
Equipment
Nanodrop™ One Spectrophotometer	Thermo-Scientific	ND-ONE-W
Refrigerated microcentrifuge Sigma 1-14K, suitable for centrifugation of 1.5 ml microcentrifuge tubes at 14,000 rpm	Sigma-Aldrich	1-14K
Disinfected adjustable pipettes, range 2-20 µl, 20-200 µl	Gilson	SKU:F167360
Disposable 1.5 ml microcentrifuge tubes (autoclaved)	Axygen	MCT-150-SP
Disposable 600 µl microcentrifuge tubes (autoclaved)	Axygen	3208
Disposable Pipette tips 1-10 µl	Axygen	T-300
Disposable Pipette tips 1-200 µl	Axygen	TR-222-Y
Dot-Blot apparatus Bio-Dot	BIORAD	1706545
Portable Hergom Suction	Hergom	7E-A
Scientific Light Box (Visible-light PH90-115V)	Hoefer	PH90-115V
UV Crosslinker	Hoefer	UVC-500
Thermo Hybaid PCR Express Thermocycler	Hybaid	HBPX110
Radiographic cassette with IP Plate14 X 17	Fuji