Name: लेखक स्पॉटलाइट: पर्यावरण विश्लेषण और रोग निगरानी के लिए पानी में लेप्टोस्पाइरा डीएनए का पता लगाना
Uploaded: 2024-06-14T12:30:00
Description: The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by ...

हम माइक्रोबायोलॉजी और इम्यूनोलॉजी विभाग, पशु चिकित्सा और ज़ूटेक्निक्स संकाय, मेक्सिको के राष्ट्रीय स्वायत्त विश्वविद्यालय के लेप्टोस्पाइरा संग्रह के ऋणी हैं। हम संदर्भ लेप्टोस्पाइरा उपभेदों के उदार दान के लिए आभारी हैं; लेप्टोस्पाइरा फेनी सेरोवर हर्स्टब्रिज स्ट्रेन BUT6 और लेप्टोस्पाइरा बिफ्लेक्सा सेरोवर पैटोक I से डॉ. एलेजांद्रो डे ला पेना मोक्टेज़ुमा। हम डॉ. जोस एंटोनियो ओकाम्पो सर्वेंटेस, CIBAC समन्वयक और कर्मियों को उनके रसद समर्थन के लिए धन्यवाद देते हैं। ईडीटी मेट्रोपॉलिटन ऑटोनॉमस यूनिवर्सिटी-कैंपस कुआजिमल्पा के स्नातक छात्रों के लिए टर्मिनल प्रोजेक्ट कार्यक्रम के तहत था। हम आंकड़े 1, और 3 से 9 के निर्माण के लिए Biorender.com सॉफ्टवेयर को स्वीकार करते हैं।

1. नमूना तैयार करना
<ol><li>4 डिग्री सेल्सियस पर 10 मिन के लिए 8,000 x ग्राम पर सेंट्रीफ्यूजेशन द्वारा 1.5 एमएल माइक्रोसेंट्रीफ्यूज ट्यूबों में प्रत्येक पानी के नमूने को केंद्रित करें। इस चरण को दोहराएं,...

पानी के नमूनों में लेप्टोस्पाइरा डीएनए का पता लगाने के लिए डॉट-ब्लॉट परख

डॉट-ब्लॉट तकनीक के महत्वपूर्ण चरणों में शामिल हैं (1) डीएनए स्थिरीकरण, (2) गैर-समरूप डीएनए के साथ झिल्ली पर मुक्त बाध्यकारी साइटों को अवरुद्ध करना, (3) एनीलिंग शर्तों के तहत जांच और लक्ष्य टुकड़े के बीच पूरक?...

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>REAGENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Purelink DNA extraction kit</td>
			<td>Invitrogen</td>
			<td>K182002</td>
			<td></td>
		</tr>
		<tr>
			<td>Gotaq Flexi DNA Polimerase (End-Point PCR Taq polymerase kit)</td>
			<td>Promega</td>
			<td>M3001</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman filter paper, grade 1,</td>
			<td>Merk</td>
			<td>WHA1001325</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Membranes, positively charged Roll 30cm x 3 m</td>
			<td>Roche</td>
			<td>11417240001</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin-AP, Fab fragments Sheep Polyclonal Primary-antibody</td>
			<td>Roche</td>
			<td>11093274910</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium Base EMJH</td>
			<td>Difco</td>
			<td>S1368JAA</td>
			<td></td>
		</tr>
		<tr>
			<td>Leptospira Enrichment EMJH</td>
			<td>Difco</td>
			<td>BD 279510</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking Reagent</td>
			<td>Roche</td>
			<td>11096176001</td>
			<td></td>
		</tr>
		<tr>
			<td>CSPD ready to use Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2&#8242;-(5&#8242;-chloro) tricyclo [3.3.1.13,7] decan}8-4-yl) phenyl phosphate</td>
			<td>Merk</td>
			<td>11755633001</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonucleic acid from herring sperm</td>
			<td>Sigma Aldrich</td>
			<td>D3159</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer Carestream</td>
			<td>Carestream Health Inc</td>
			<td>GBX5158621</td>
			<td></td>
		</tr>
		<tr>
			<td>Digoxigenin-11-ddUTP</td>
			<td>Roche</td>
			<td>11363905910</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, Disodium Salt (Dihydrate)</td>
			<td>Promega</td>
			<td>H5032</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400</td>
			<td>Sigma Aldrich</td>
			<td>F8016</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixer Carestream</td>
			<td>Carestream Health Inc</td>
			<td>GBX 5158605</td>
			<td></td>
		</tr>
		<tr>
			<td>Lauryl sulfate Sodium Salt (Sodium dodecyl sulfate; SDS) C12H2504SNa</td>
			<td>Sigma Aldrich</td>
			<td>L5750</td>
			<td></td>
		</tr>
		<tr>
			<td>N- Lauroylsarcosine sodium salt CH3(CH2)10CON(CH3) CH2COONa</td>
			<td>Sigma Aldrich</td>
			<td>L-9150</td>
			<td>It is an anionic surfactant</td>
		</tr>
		<tr>
			<td>Polivinylpyrrolidone (PVP-40)</td>
			<td>Sigma Aldrich</td>
			<td>PVP40</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol Sorbitan monolaurate (Tween 20)</td>
			<td>Sigma Aldrich</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma Aldrich</td>
			<td>151-21-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma Aldrich</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (NaH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>7558-79-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Terminal transferase, recombinant</td>
			<td>Roche</td>
			<td>3289869103</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride (Tris HCl)</td>
			<td>Sigma-Aldrich</td>
			<td>1185-53-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SSPE 20X</td>
			<td>Sigma-Aldrich</td>
			<td>S2015-1L</td>
			<td>It can be Home-made following Supplementary File 6</td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Sigma-Aldrich</td>
			<td>On demand</td>
			<td>Follow table 1</td>
		</tr>
		<tr>
			<td>Probes</td>
			<td>Sigma-Aldrich</td>
			<td>On demand</td>
			<td>Follow table 1</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop&#8482; One Spectrophotometer</td>
			<td>Thermo-Scientific</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated microcentrifuge Sigma 1-14K, suitable for centrifugation of 1.5 ml microcentrifuge tubes at 14,000 rpm</td>
			<td>Sigma-Aldrich</td>
			<td>1-14K</td>
			<td></td>
		</tr>
		<tr>
			<td>Disinfected adjustable pipettes, range 2-20 &#181;l, 20-200 &#181;l</td>
			<td>Gilson</td>
			<td>SKU:F167360</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 1.5 ml microcentrifuge tubes (autoclaved)</td>
			<td>Axygen</td>
			<td>MCT-150-SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 600 &#181;l microcentrifuge tubes (autoclaved)</td>
			<td>Axygen</td>
			<td>3208</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Pipette tips 1-10 &#181;l</td>
			<td>Axygen</td>
			<td>T-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Pipette tips 1-200 &#181;l</td>
			<td>Axygen</td>
			<td>TR-222-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>Dot-Blot apparatus Bio-Dot</td>
			<td>BIORAD</td>
			<td>1706545</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable Hergom Suction</td>
			<td>Hergom</td>
			<td>7E-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scientific Light Box (Visible-light PH90-115V)</td>
			<td>Hoefer</td>
			<td>PH90-115V</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Crosslinker</td>
			<td>Hoefer</td>
			<td>UVC-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Hybaid PCR Express Thermocycler</td>
			<td>Hybaid</td>
			<td>HBPX110</td>
			<td></td>
		</tr>
		<tr>
			<td>Radiographic cassette with IP Plate14 X 17</td>
			<td>Fuji</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

polymerase chain reaction and dot-blot hybridization for leptospira detection in water samples

डॉट-ब्लॉट एक सरल, तेज, संवेदनशील और बहुमुखी तकनीक है जो विशेष रूप से वाहक डीएनए की उपस्थिति में जांच संकरण द्वारा लक्षित डीएनए की न्यूनतम मात्रा की पहचान करने में सक्षम बनाती है। यह एक अक्रिय ठोस समर्थन पर डीएनए की एक ज्ञात मात्रा के हस्तांतरण पर आधारित है, जैसे कि नायलॉन झिल्ली, डॉट-ब्लॉट तंत्र का उपयोग करके और इलेक्ट्रोफोरेटिक पृथक्करण के बिना। नायलॉन झिल्ली में उच्च न्यूक्लिक एसिड बाध्यकारी क्षमता (400 μg / सेमी2), उच्च शक्ति का लाभ होता है, और सकारात्मक या तटस्थ रूप से चार्ज किया जाता है। उपयोग की जाने वाली जांच 18 से 20 आधारों का एक अत्यधिक विशिष्ट ssDNA टुकड़ा है जो लंबे समय तक digoxigenin (DIG) के साथ लेबल किया गया है। जांच लेप्टोस्पाइरा डीएनए के साथ संयुग्मित होगी। एक बार जब जांच लक्ष्य डीएनए के साथ संकरित हो जाती है, तो यह एक एंटी-डिगॉक्सिजेनिन एंटीबॉडी द्वारा पता लगाया जाता है, जिससे एक्स-रे फिल्म में प्रकट इसके उत्सर्जन के माध्यम से इसका आसान पता लगाया जा सकता है। उत्सर्जन के साथ डॉट्स ब्याज के डीएनए टुकड़ों के अनुरूप होंगे। यह विधि जांच के गैर-आइसोटोपिक लेबलिंग को नियोजित करती है, जिसमें बहुत लंबा आधा जीवन हो सकता है। इस मानक इम्यूनो-लेबल का दोष आइसोटोपिक जांच की तुलना में कम संवेदनशीलता है। फिर भी, यह युग्मन पोलीमरेज़ चेन रिएक्शन (पीसीआर) और डॉट-ब्लॉट परख द्वारा कम किया जाता है। यह दृष्टिकोण लक्ष्य अनुक्रम के संवर्धन और इसकी पहचान को सक्षम बनाता है। इसके अतिरिक्त, यह एक मात्रात्मक अनुप्रयोग के रूप में इस्तेमाल किया जा सकता है जब एक प्रसिद्ध मानक के एक धारावाहिक कमजोर पड़ने के खिलाफ तुलना की जाती है। पानी के नमूनों में तीन मुख्य समूहों से लेप्टोस्पाइरा का पता लगाने के लिए एक डॉट-ब्लॉट एप्लिकेशन यहां प्रस्तुत किया गया है। लेप्टोस्पाइरल डीएनए की उपस्थिति का प्रमाण प्रदान करने के लिए सेंट्रीफ्यूजेशन द्वारा केंद्रित होने के बाद इस पद्धति को बड़ी मात्रा में पानी पर लागू किया जा सकता है। यह सामान्य स्क्रीनिंग उद्देश्यों के लिए एक मूल्यवान और संतोषजनक उपकरण है, और इसका उपयोग अन्य गैर-संवर्धन योग्य बैक्टीरिया के लिए किया जा सकता है जो पानी में मौजूद हो सकते हैं, पारिस्थितिकी तंत्र की समझ को बढ़ाते हैं।

Leptospirosis in humans mainly originates from environmental sources1,2. 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 water1,3,4. Furthermore, Leptospira has been identified in non-natural sources, including sewage, stagnant and tap water5,6.
Leptospira is a worldwide distributed bacteria7,8, 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 conditions12,13.&#160;It may be part of biofilms in freshwater with Azospirillum and Sphingomonas and is even capable of growing and enduring temperatures exceeding 49 &#176;C14,15. 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 year17,18. 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-blot24,25,26.
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 &#956;g/cm2 of nucleic acids and up to 400 &#956;g/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 clades30,31. The distinction among these clades can be achieved based on specific genes such as lipL41, lipL32, and the 16S&#160;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&#160;known for its stable expression and used in molecular techniques32, while the 16S&#160;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,&#160;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&#176; 16&#39; 54&#34; N 99&#176; 6&#39; 11&#34; 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 &#176;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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65435/65435fig01v2.jpg" /> 
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). <a href="https://www.jove.com/files/ftp_upload/65435/65435fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
DNA extraction 
Total DNA was isolated using a commercial Genomic DNA kit according to the manufacturer&#39;s instructions (see Table of Materials). DNA extractions were eluted in 20 &#181;L of elution buffer, and DNA concentration was determined by a UV Spectrophotometer at 260-280 nm, and stored at 4 &#176;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. <a href="https://www.jove.com/files/ftp_upload/65435/TABLE_1_ 7th_PR_ EMCC.xlsx" target="_blank">Please click here to download this Table.</a>
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&#39; end (Digoxigenin is a plant steroid obtained from Digitalis purpurea, used&#160;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).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65435/65435fig02v2.jpg" /> 
Figure 2: Steps of the procedure for the PCR-dot-blot assay. <a href="https://www.jove.com/files/ftp_upload/65435/65435fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

लेखक स्पॉटलाइट: पर्यावरण विश्लेषण और रोग निगरानी के लिए पानी में लेप्टोस्पाइरा डीएनए का पता लगाना

पोलीमरेज़ चेन रिएक्शन और पानी के नमूनों में लेप्टोस्पाइरा का पता लगाने के लिए डॉट-ब्लॉट हाइब्रिडाइजेशन

This protocol is designed to identify the presence of Leptospira DNA in water samples. The dot-blot technique is utilized for this purpose, which enables accurate detection and analysis of the DNA. The dot-blot is an easy and versatile non-isotopic technique and may help to answer key questions about the environmental distribution of Leptospira.This method has the potential to assess huge water bodies and aid in disease surveillance.

तकनीक की प्रभावशीलता का आकलन करने के लिए, प्रत्येक लेप्टोस्पाइरा सेरोवर की शुद्ध संस्कृतियों से जीनोमिक डीएनए का उपयोग क्लेड-विशिष्ट जांच के साथ किया गया था। झिल्ली प्रत्येक सेरोवर के लिए पीसीआर ...

इस अध्ययन में, पानी के नमूनों में तीन मुख्य समूहों से लेप्टोस्पाइरा का पता लगाने के लिए एक डॉट-ब्लॉट एप्लिकेशन डिज़ाइन किया गया था। यह विधि विशेष रूप से एक डिगॉक्सिजेनिन-लेबल जांच द्वारा लक्षित न्यूनतम डीएनए मात्रा की पहचान के लिए अनुमति देती है, आसानी से एक एंटी-डिगॉक्सिजेनिन एंटीबॉडी द्वारा पता लगाया जाता है। यह दृष्टिकोण स्क्रीनिंग उद्देश्यों के लिए एक मूल्यवान और संतोषजनक उपकरण है।

The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by ...

यह प्रोटोकॉल पानी के नमूनों में लेप्टोस्पाइरा डीएनए की उपस्थिति की पहचान करने के लिए डिज़ाइन किया गया है। इस उद्देश्य के लिए डॉट-ब्लॉट तकनीक का उपयोग किया जाता है, जो डीएनए का सटीक पता लगाने और विश्लेषण करने में सक्षम बनाता है। डॉट-ब्लॉट एक आसान और बहुमुखी गैर-आइसोटोपिक तकनीक है और लेप्टोस्पाइरा के पर्यावरणीय वितरण के बारे में महत्वपूर्ण सवालों के जवाब देने में मदद कर सकती है।इस पद्धति में विशाल जल निकायों का आकलन करने और रोग निगरानी में सहायता करने की क्षमता है।

polymerase-chain-reaction-dot-blot-hybridization-for-leptospira

Research

JoVE Journal

Biology

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

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 &#181;g/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.

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.