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.

Le....

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....

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