Zobraziť v EDS Plný text vo formáte HTML Plný text ve formátu PDF

Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) for Rapid Diagnosis of Newcastle Disease in Field and Resource-Limited Areas.

Uložené v:
Podrobná bibliografia
Názov: Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) for Rapid Diagnosis of Newcastle Disease in Field and Resource-Limited Areas.
Autori: Mihiretu, Berihun Dires (AUTHOR), Usui, Tatsufumi (AUTHOR), Chibssa, Tesfaye Rufael (AUTHOR), Mohammed, Abde Aliy (AUTHOR), Yamaguchi, Tsuyoshi (AUTHOR)
Zdroj: Veterinary Medicine: Research & Reports. Nov2025, Vol. 16, p39-47. 9p.
Druh dokumentu: Article
Predmet: Newcastle disease, Newcastle disease virus, Diagnosis, Molecular diagnosis, Poultry, Loop-mediated isothermal amplification
Author-Supplied Keywords: loop mediated isothermal amplification
RNA preparation
Abstrakt: Background: Newcastle Disease (ND) is a highly contagious and fatal poultry disease caused by Newcastle disease virus (NDV) and has a global distribution. To control this disease, a rapid method for diagnosing it is needed. PCR-based molecular diagnostics such as real-time reverse transcription-PCR (RRT-PCR) for detecting NDV genes have been conducted worldwide. However, because PCR methods are time-consuming and require a well-equipped laboratory, they are not well-suited for use in field and resource limited areas. Methods: In this study, we established a field-friendly loop-mediated isothermal amplification (LAMP) assay for NDV diagnosis using primers targeting the highly conserved L gene, which bypasses the need for RNA extraction and makes it more suitable for field application than previously reported LAMP methods. Results: Our LAMP method is capable of detecting a broad range of NDV genotypes and showed no cross-reactivity with other avian viral diseases or the host genome. The reaction is completed within 35 minutes of incubation at 65°C. NDV was successfully detected directly from swab and tissue samples without the need for conventional RNA extraction. Conclusion: The LAMP method developed in this study offers a rapid, affordable, and field-friendly diagnostic tool for NDV detection. [ABSTRACT FROM AUTHOR]
Copyright of Veterinary Medicine: Research & Reports is the property of Dove Medical Press Ltd and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
Full Text Word Count: 4246
ISSN: 2230-2034
DOI: 10.2147/VMRR.S549952
Prístupové číslo: 190276331
Databáza: Veterinary Source
Neprihláseným uživateľom se plný text nezobrazuje
FullText Links:
  – Type: pdflink
    Url: https://content.ebscohost.com/cds/retrieve?content=AQICAHjPtM4BHU3ZchRwgzYmadcigk49r9CVlbU7V5F6lgH7WwHXWFGgdr60diMG4WaQnYnvAAAA4zCB4AYJKoZIhvcNAQcGoIHSMIHPAgEAMIHJBgkqhkiG9w0BBwEwHgYJYIZIAWUDBAEuMBEEDG3BIcJlmJq3nUZrbQIBEICBm49-_SIGZL4xx4z6_wya1CUyWdcpDCy3z3srHambh9agyXqFyIFFF5WWLK-0D59AanKnDfyLAb5VZJxmal10S3c5CgjwhK5DZySbrbZer0K31izsW-DpeTsFNNeQQyACfvZXitdP1ukw8GyufGTAaY68O0yxfJkL6-EtcGhNOHn9pkXgiuVFQAtJ6ZeTTvtWnof-MeFcz5YQO3HW
Text:
  Availability: 1
  Value: <anid>AN0190276331;[eh5d]01nov.25;2025Dec18.03:50;v2.2.500</anid> <title id="AN0190276331-1">Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) for Rapid Diagnosis of Newcastle Disease in Field and Resource-Limited Areas </title> <p>Background: Newcastle Disease (ND) is a highly contagious and fatal poultry disease caused by Newcastle disease virus (NDV) and has a global distribution. To control this disease, a rapid method for diagnosing it is needed. PCR-based molecular diagnostics such as real-time reverse transcription-PCR (RRT-PCR) for detecting NDV genes have been conducted worldwide. However, because PCR methods are time-consuming and require a well-equipped laboratory, they are not well-suited for use in field and resource limited areas. Methods: In this study, we established a field-friendly loop-mediated isothermal amplification (LAMP) assay for NDV diagnosis using primers targeting the highly conserved L gene, which bypasses the need for RNA extraction and makes it more suitable for field application than previously reported LAMP methods. Results: Our LAMP method is capable of detecting a broad range of NDV genotypes and showed no cross-reactivity with other avian viral diseases or the host genome. The reaction is completed within 35 minutes of incubation at 65°C. NDV was successfully detected directly from swab and tissue samples without the need for conventional RNA extraction. Conclusion: The LAMP method developed in this study offers a rapid, affordable, and field-friendly diagnostic tool for NDV detection.</p> <p>Keywords: loop mediated isothermal amplification; Newcastle disease; RNA preparation</p> <p>Berihun Dires Mihiretu,<sups>1</sups> Tatsufumi Usui,<sups>1,</sups><sups>2</sups> Tesfaye Rufael Chibssa,<sups>3</sups> Abde Aliy Mohammed,<sups>3</sups> Tsuyoshi Yamaguchi<sups>1,</sups><sups>2</sups></p> <p> <sups>1</sups>Joint Graduate School of Veterinary Sciences, Tottori University, Tottori, Japan; <sups>2</sups>Avian Infectious Disease and Global Health Research Center, Tottori University, Tottori, Japan; <sups>3</sups>Molecular Biology Laboratory, Animal Health Institute, Sebeta, Ethiopia</p> <p>Correspondence: Tsuyoshi Yamaguchi, Avian Infectious Disease and Global Health Research Center, Tottori University, Koyama-cho Minami 4-101, Tottori, 680-8553, Japan, Tel/Fax +81-857-31-5487, Email [email protected]</p> <hd id="AN0190276331-2">Introduction</hd> <p>Newcastle disease (ND) is a highly contagious and fatal disease of poultry caused by Newcastle disease virus (NDV), a negative-sense single-stranded RNA virus. NDV has six genes that encode six structural proteins: nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and large protein (L), in the 3′ to 5′ direction.[<reflink idref="bib1" id="ref1">1</reflink>] NDV is classified into two major classes: Class I and Class II. Class I NDVs are genetically less diverse and consist of a single genotype, whereas Class II NDVs are more diverse and have been classified into 21 genotypes based on the fusion (F) gene sequences.[<reflink idref="bib2" id="ref2">2</reflink>] Based on the clinical signs observed in infected chickens, NDV strains are further classified into five pathotypes: Viscerotropic velogenic: Highly virulent, causing intestinal hemorrhages, Neurotropic velogenic: Highly virulent, associated with respiratory and neurological symptoms, Mesogenic: Moderately virulent, causing respiratory signs and occasional nervous symptoms, Lentogenic: Low virulence, result in mild respiratory infections and subclinical: infections without apparent symptoms.[<reflink idref="bib3" id="ref3">3</reflink>]</p> <p>Rapid diagnosis of NDV is important to prevent the spread of the disease. Real-time reverse transcription polymerase chain reaction (RRT-PCR), targeting the M gene,[<reflink idref="bib4" id="ref4">4</reflink>] is the most commonly used diagnostic method. However, this technique is labor-intensive, expensive, and requires well-equipped laboratories. An excellent alternative to RRT-PCR is loop-mediated isothermal amplification (LAMP), a method based on strand displacement reaction developed by Notomi et al.[<reflink idref="bib5" id="ref5">5</reflink>] LAMP is performed at a constant temperature and does not require a thermal cycler. It utilizes four to six primers, allowing for highly specific and sensitive amplification of the target gene.[<reflink idref="bib5" id="ref6">5</reflink>],[<reflink idref="bib6" id="ref7">6</reflink>] LAMP results can be obtained in a short time, and interpretation can be made visually, without the need for additional equipment or complex procedures.[<reflink idref="bib7" id="ref8">7</reflink>]</p> <p>LAMP-based methods have been developed for NDV diagnosis.8–12 With the exception of Song et al,[<reflink idref="bib12" id="ref9">12</reflink>] all of these methods were based on the F gene. World Organization for Animal Health (WOAH),[<reflink idref="bib3" id="ref10">3</reflink>] recommends that molecular screening of NDV be based on conserved genes such as the matrix (M) and large polymerase (L) genes. Since the F gene is one of the most diverse NDV genes used for genotyping,[<reflink idref="bib2" id="ref11">2</reflink>] F gene-based LAMP primers may not effectively detect the broad range of NDV genotypes currently circulating worldwide. Song et al[<reflink idref="bib12" id="ref12">12</reflink>] developed a LAMP method targeting the F (for virulent NDVs) and HN gene (for all NDV strains). However, the HN is also less conserved,[<reflink idref="bib3" id="ref13">3</reflink>] which may limit the assay's specificity and broad-range detection capability. Moreover, all of these approaches depend on RNA extracted using conventional RNA extraction kits, which can be expensive, particularly in resource-limited settings. In recent years, following the global COVID-19 pandemic, several new techniques have been reported that enable the preparation of RNA for the detection of viral genes from clinical materials in a simple manner without the use of special kits or equipment.13–15 To enable NDV testing in the field and in resource-limited settings, a more affordable and easily applicable method is needed. Therefore, the objective of this study was to develop a user and field-friendly LAMP assay by eliminating the RNA extraction step and to design primers targeting the L gene, one of the most conserved regions of the NDV genome,[<reflink idref="bib3" id="ref14">3</reflink>] to allow the detection of a broad range of NDV genotypes.</p> <hd id="AN0190276331-3">Materials and Methods</hd> <p></p> <hd id="AN0190276331-4">Designing Primers</hd> <p>LAMP primers were designed from the large polymerase (L) gene of NDVs. In order to design a primer that can detect a wide range of NDV pathotypes, consensus sequences were made from 19 NDV genotypes downloaded from GenBank (Table 1). The sequences were aligned by MUltiple Sequence Comparison by Log-Expectation (MUSCLE) using MEGA11.[<reflink idref="bib16" id="ref15">16</reflink>] The consensus sequences were viewed by Jalview[<reflink idref="bib17" id="ref16">17</reflink>] and six LAMP primers were designed using the NEB LAMP primer design tool (https://lamp.neb.com/#!/). The designed primers included: Forward inner primer (FIP), Backward inner primer (BIP), Forward outer primer (F3), Backward outer primer (B3), Forward loop primer (FL) and Backward loop primer (LB).</p> <p></p> <p> <ephtml> <table><tbody><tr><td><p><graphic href="article/fulltext_file/549952/aW1n/VMRR_A_549952_t0001_Thumb.jpg" content-type="Graph" /></p></td><td><p><bold>Table 1</bold> NDV Sequences Used for Designing LAMP Primers</p></td></tr></tbody></table> </ephtml> </p> <hd id="AN0190276331-5">RNA Preparation</hd> <p>RNAs were prepared using two different methods. The first involved a conventional RNA extraction method using the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany, Cat. No. 52906). The RNA was extracted from oropharyngeal and cloacal swab samples, as well as tissue samples of chickens, confirmed to be NDV positive by RRT-PCR using M gene primer[<reflink idref="bib4" id="ref17">4</reflink>] following the manufacturer's instructions. The extracted RNA was stored at –70°C until use.</p> <p>The second method was a modified version of the RNA extraction-free protocol described by Guan et al[<reflink idref="bib14" id="ref18">14</reflink>] for SARS-CoV-2. In addition, we incorporated 40 mM guanidine hydrochloride, which has been previously reported to enhance LAMP sensitivity for SARS-CoV-2 detection.[<reflink idref="bib18" id="ref19">18</reflink>] Briefly, swabs or homogenized tissue samples were centrifuged at 6000 rpm for 5 minutes, and the supernatant was transferred to a new tube. A 5% Chelex 100 resin (Bio-Rad, California, USA, Cat. No. 142-1253) was then added directly to the supernatant. The mixture was heated at 98°C for 5 minutes using a Dry Bath Plus (Nichiryo, Saitama, Japan, Product code 00-NDB+), followed by centrifugation at 6000 rpm for 2 minutes. The supernatant was used directly for the LAMP reaction.</p> <hd id="AN0190276331-6">LAMP Assay</hd> <p>LAMP reactions were conducted in a total volume of 12.5 μL containing 6.25 μL of Warmstart Colorimetric LAMP 2X master mix (NEB, Ipswich, MA, USA, Product No. M100S), 1.25 μL of 10X primer mix containing all 6 LAMP primers (16 μM FIP, 16 μM BIP, 2 μM F3, 2 μM B3, 4 μM LF and 4 μM LB), 0.5 μL of 1M guanidine hydrochloride (40 mM), 3.75 μL of nuclease free water and 0.75 μL of viral RNA. The reactions were carried out at 65°C for 35 minutes in Dry Bath Plus (Nichiryo, Saitama, Japan). Phenol red, a pH-sensitive dye included in the Warmstart Colorimetric LAMP 2X master mix, changes color from pink to yellow at low pH, which indicates a positive reaction.</p> <hd id="AN0190276331-7">Specificity and Sensitivity of the LAMP Assay</hd> <p>For a specificity test, RNA was extracted using a QIAamp viral RNA mini kit (Qiagen, Hilden, Germany) from avian paramyxovirus 3 (APMV-3), avian paramyxovirus 4 (APMV-4), avian paramyxovirus 6 (APMV-6) and H5N8 avian influenza virus (AIV) isolated in 9–11 day-old embryonated chicken eggs (ECE). The infectious bursal disease virus (IBDV) RNA was extracted from cloacal swab collected in viral transport medium. To assess the assay's ability to detect a broad range of NDV genotypes, RNA extracted from genotype I (Ishii), genotype II (Hitchner B1 (HB1)), and genotype IV (Herts) isolated in 9–11 day-old ECEs were tested. In addition, RNAs extracted from five chicken swabs (three tracheal, two oropharyngeal, and five cloacal) and tissues (brain and lung-trachea) of NDV-free chickens by RRT-PCR, were examined to check for any non-specific reactions with the chicken genome. To evaluate the sensitivity of the LAMP assay, 16 oropharyngeal and 10 cloacal swabs confirmed to be NDV-positive by RRT-PCR were used. Detailed profiles of these samples are provided in the previous study.[<reflink idref="bib19" id="ref20">19</reflink>] LAMP sensitivity was assessed by comparing the LAMP results with the RRT-PCR results for each tested sample.</p> <hd id="AN0190276331-8">Evaluation of LAMP Assay Using Tissue Samples</hd> <p>To evaluate the LAMP protocol under field conditions, all reagents required for the test were transported from Japan to Ethiopia and stored at the optimal temperature upon arrival at the Animal Health Institute (AHI), Sebeta, Ethiopia. The LAMP reagents were examined using RNA prepared from NDV-positive brain and lung-trachea tissue to verify whether the reagents remained unaffected by transportation at ambient temperature. These tissue samples were previously stored at –80°C at the AHI. The master mix, comprising WarmStart Colorimetric LAMP 2X master mix, 10X primer mix, guanidine hydrochloride, and nuclease-free water, was prepared in PCR tubes. For the positive control, RNA extracted from NDV antigen (Antigen NDV pigeon Batch 2/13, Inactivated, Expiry 02/2025) provided by the WOAH was added to a designated positive control tube.</p> <hd id="AN0190276331-9">Field Application of RNA Extraction-Free LAMP Assay</hd> <p>The prepared master mix was transported on ice to a poultry farm in Sebeta, Ethiopia. Supporting equipment including a Dry Bath Plus, a portable vortex mixer, and a centrifuge were also brought to the site. Oropharyngeal and cloacal swab samples were collected from two chickens and tested on-site using the LAMP assay at 65°C for 35 minutes.</p> <hd id="AN0190276331-10">Results</hd> <p></p> <hd id="AN0190276331-11">Optimization of LAMP Method</hd> <p>LAMP primers targeting conserved regions of the NDV L gene were designed and screened for their ability to specifically detect NDV RNA without non-specific amplification. To optimize assay conditions, the LAMP reaction was tested at temperatures ranging from 60°C to 67°C. Initial screening was conducted using RNA extracted from the lentogenic HB1 vaccine strain and two velogenic genotype VII.1.1 NDV isolates from Ethiopian chickens (NDV/chicken/Eth_HT9C15/2023 and NDV/chicken/Eth_SLBM12C1/2023,[<reflink idref="bib19" id="ref21">19</reflink>] all propagated in 9–11 day-old ECEs. The LAMP primers shown in Table 2 were selected for our LAMP method. The optimal reaction condition was established as incubation at 65°C for 35 minutes.</p> <p></p> <p> <ephtml> <table><tbody><tr><td><p><graphic href="article/fulltext_file/549952/aW1n/VMRR_A_549952_t0002_Thumb.jpg" content-type="Graph" /></p></td><td><p><bold>Table 2</bold> LAMP Primer Set Based on NDV L Gene</p></td></tr></tbody></table> </ephtml> </p> <hd id="AN0190276331-12">Specificity of the LAMP Method</hd> <p>Once positive results were obtained using RNA extracted from the HB1 strain and the two velogenic NDV isolates (Figure 1a), the specificity of the LAMP method was evaluated. RNAs from IBDV, APMV-3, APMV-4, APMV-6, and H5N8 AIV were tested, and no non-specific amplifications were observed (Figure 1b). The LAMP method developed in this study successfully detected all tested NDV genotypes: genotype I (Ishii), II (HB1), and IV (Herts) within 35 minutes of incubation (Figure 1c). To evaluate the specificity of the RNA extraction-free method, the LAMP assay was tested for potential non-specific amplification against chicken genomic material and swab samples. Tracheal, oropharyngeal, and cloacal swabs were collected from five NDV-free chickens and used in RNA extraction-free LAMP reactions. No non-specific amplification was observed in any of the samples.</p> <p></p> <p> <ephtml> <table><tbody><tr><td><p><graphic href="article/fulltext_file/549952/aW1n/VMRR_A_549952_O_F0001g_Thumb.jpg" content-type="Graph" /></p></td><td><p><bold>Figure 1</bold> Specificity of the LAMP method. (<bold>a</bold>) Successful amplification was observed using RNA extracted from two velogenic NDV strains (1 and 2) and the lentogenic HB1 strain (3); (<bold>b</bold>) No amplification was detected for other avian viral pathogens: IBDV (1), APMV-3 (2), APMV-4 (3), APMV-6 (4), and H5N8 AIV (5); (<bold>c</bold>) LAMP test results for NDV genotype I (1), II (2), and IV (3). Velogenic NDV strains used: NDV/chicken/Eth_HT9C15/2023 (Accession number: LC846631) and NDV/chicken/Eth_SLBM12C1/2023 (Accession number: LC846618). Negative control (NC): nuclease-free water. A positive reaction is indicated by a color change from pink to yellow.</p></td></tr></tbody></table> </ephtml> </p> <hd id="AN0190276331-13">Sensitivity of LAMP Method</hd> <p>Sensitivity of our LAMP method was compared with RRT-PCR by conducting LAMP reactions using RNA extracted with a QIAamp viral RNA mini kit (Qiagen, Hilden, Germany) (purified RNA). The results of the LAMP method were 100% consistent with those of RRT-PCR for all 16 oropharyngeal swabs (Ct values: 23.09–33.1) and 10 cloacal swabs (Ct values: 22.97–32.02) (Figures 2a, b and 3a), resulting in 100% sensitivity and positive predictive value. All samples prepared using the RNA extraction-free method with Ct values ≤ 31.3 were successfully detected by RRT-PCR (Figure 3b). Furthermore, samples prepared using an RNA extraction-free method (unpurified RNA) were tested by RRT-PCR to assess their suitability. The unpurified RNA Ct values were increased by 2–9 cycles for oropharyngeal swabs and 4–6 cycles for cloacal swabs (Figure 3c).</p> <p></p> <p> <ephtml> <table><tbody><tr><td><p><graphic href="article/fulltext_file/549952/aW1n/VMRR_A_549952_O_F0002g_Thumb.jpg" content-type="Graph" /></p></td><td><p><bold>Figure 2</bold> LAMP test results using extracted RNA for NDV-positive oropharyngeal and cloacal swab clinical samples. (<bold>a</bold>) Sixteen oropharyngeal swabs (1–16); (<bold>b</bold>) Ten cloacal swabs (1–10). A positive reaction is indicated by a color change from pink to yellow.</p><p><bold>Abbreviation</bold>: NC, negative control.</p></td></tr></tbody></table> </ephtml> </p> <p></p> <p> <ephtml> <table><tbody><tr><td><p><graphic href="article/fulltext_file/549952/aW1n/VMRR_A_549952_O_F0003g_Thumb.jpg" content-type="Graph" /></p></td><td><p><bold>Figure 3</bold> Comparison of LAMP and RRT-PCR assays. (<bold>a</bold>) Results of LAMP and RRT-PCR using RNA extracted from 16 oropharyngeal and 10 cloacal swab samples; (<bold>b</bold>) Results of LAMP and RRT-PCR using the same samples prepared by an RNA extraction-free method; (<bold>c</bold>) Comparison of Ct values obtained using extracted RNA and RNA extraction-free preparation methods. Filled circles (●): Oropharyngeal swabs, open circles (○): Cloacal swabs.</p></td></tr></tbody></table> </ephtml> </p> <hd id="AN0190276331-14">LAMP Test for Clinical Samples Using RNA Extraction-Free Method</hd> <p>A total of 16 NDV-positive oropharyngeal swabs with cycle threshold (Ct) values ranging from 23.09 to 33.1 and 10 NDV-positive cloacal swabs with Ct values ranging from 22.97 to 32.02, as determined by RRT-PCR, were processed using an RNA extraction-free method (unpurified RNA). All of these sample were previously classified as sub-genotype VII.1.1 of genotype VII NDVs.[<reflink idref="bib19" id="ref22">19</reflink>] Among samples with Ct values ≤ 25, 83.3% (5/6) of oropharyngeal swabs (Or1-6) and 100% (2/2) of cloacal swabs (C1-2) were successfully detected using the rapid and user-friendly LAMP assay developed in this study, resulting in 87.5% sensitivity and 100% positive predictive value. In contrast, for samples with Ct values > 25, only 10% (1/10) of oropharyngeal swabs (Or7-16) and 25% (2/8) of cloacal swabs (C3-10) were detected by the LAMP method (Figures 3b and 4a, b).</p> <p></p> <p> <ephtml> <table><tbody><tr><td><p><graphic href="article/fulltext_file/549952/aW1n/VMRR_A_549952_O_F0004g_Thumb.jpg" content-type="Graph" /></p></td><td><p><bold>Figure 4</bold> LAMP test results using RNA extraction-free method for NDV-positive oropharyngeal and cloacal swab clinical samples. (<bold>a</bold>) Sixteen oropharyngeal swabs (1–16); (<bold>b</bold>) Ten cloacal swabs (1–10). A positive reaction is indicated by a color change from pink to yellow.</p><p><bold>Abbreviation</bold>: NC, negative control.</p></td></tr></tbody></table> </ephtml> </p> <hd id="AN0190276331-15">Evaluation of LAMP Assay Using Tissue Samples</hd> <p>The LAMP method, tested at the AHI in Ethiopia using samples prepared with an RNA extraction-free method from NDV-positive brain and lung-trachea tissues, showed positive results. No amplification was detected in NDV-negative brain and lung-trachea tissues, which were used as controls to evaluate any potential reactivity against host tissues (Figure 5a). Furthermore, the LAMP test on RNA extracted from six NDV-positive brain and lung samples, with Ct values ranging from 24.39 to 29.06, demonstrated 100% positivity, consistent with the results obtained using real-time RT-PCR (RRT-PCR) (Figure 5b).</p> <p></p> <p> <ephtml> <table><tbody><tr><td><p><graphic href="article/fulltext_file/549952/aW1n/VMRR_A_549952_O_F0005g_Thumb.jpg" content-type="Graph" /></p></td><td><p><bold>Figure 5</bold> LAMP assay for field application: (<bold>a</bold>) LAMP test results using RNA extraction-free method from NDV-positive (1, 2) and NDV-negative (3, 4) brain and lung-trachea tissues; (<bold>b</bold>) LAMP test results using extracted RNA for NDV-positive brain (2, 4, 5) and lung-trachea (1, 3) tissues with Ct values ranging from 24.39 to 29.06; (<bold>c</bold>) Equipment and reagents transported to the field; (<bold>d</bold>) Sample collection in the field; (<bold>e</bold>) LAMP assay using an RNA extraction-free method conducted on-site; (<bold>f</bold>) LAMP test results using an RNA extraction-free method at the field, with oropharyngeal (1, 2) and cloacal (3, 4) swabs. PC: positive control (RNA from NDV antigen); NC: negative control (nuclease-free water).</p></td></tr></tbody></table> </ephtml> </p> <hd id="AN0190276331-16">Field Application of LAMP Assay</hd> <p>For the field application, the master mix prepared in the laboratory and transported to the poultry farm produced a positive result only for the NDV-positive control. The two oropharyngeal and two cloacal swabs collected and tested on-site were negative by the LAMP method (Figure 5c–f). These swab samples were also confirmed to be NDV-negative by RRT-PCR using RNA extracted from the same specimens.</p> <hd id="AN0190276331-17">Discussion</hd> <p>ND is a major problem in poultry production, particularly in developing countries where diagnostic laboratories and infrastructures are limited. Rapid diagnosis of NDV is essential to provide effective control measures. However, the most commonly used diagnostic method, RRT-PCR requires expensive equipment and well-established laboratory settings. In this study, we developed a rapid, cost-effective, and easily applicable diagnostic method for NDV that can be used in the field and in resource-limited settings. The LAMP assay, combined with an RNA extraction-free approach, enables direct detection of NDV from swab and tissue samples, without the need for conventional RNA extraction kits. Moreover, the reaction can be performed using a portable heat block, making it highly suitable for field diagnosis, with results obtained within 35 minutes of incubation at a constant temperature of 65°C. We have confirmed suitability of the method through field testing. This LAMP method effectively detects NDV in samples with Ct values ≤ 25, making it practical for field diagnosis in NDV outbreaks where the viral load is expected to be high. Samples prepared using the RNA extraction-free method can also be used for RRT-PCR in laboratories where RNA extraction kits are limited.</p> <p>Our LAMP method demonstrates high specificity and sensitivity for NDV. No cross-reaction was observed with other avian viral diseases tested in this study (IBDV, AMPV-3, APMV-4, APMV-6, and H5N8 AIV), and no non-specific amplification was detected against the host genome. Furthermore, our LAMP assay successfully detected a broad range of NDV genotypes, including both lentogenic and velogenic pathotypes. This makes it a viable alternative to the commonly used M-gene-based RRT-PCR[<reflink idref="bib4" id="ref23">4</reflink>] for diagnosing all NDV pathotypes. The sensitivity of our LAMP method is 100% consistent with RRT-PCR when using RNA extracted with a commercial RNA extraction kit. These results suggest that the LAMP method established in this study can be a reliable alternative to RRT-PCR for the broad detection of NDV genotypes across all pathotypes.</p> <p>Previously established LAMP methods for NDV diagnosis have some limitations. The method developed by Pham et al[<reflink idref="bib10" id="ref24">10</reflink>] requires cDNA synthesis and a reaction time of 2 hours. Additionally, Li et al[<reflink idref="bib9" id="ref25">9</reflink>] reported that the primers designed by Pham et al[<reflink idref="bib10" id="ref26">10</reflink>] resulted in false-negative results. To address this, Li et al[<reflink idref="bib9" id="ref27">9</reflink>] developed a one-step reverse transcription (RT)-LAMP method based on F gene primers. However, the visualization method used in this assay requires the addition of magnesium ions or SYBR Green I, necessitating the opening of the reaction tube, which increases the risk of laboratory contamination. Kirunda et al[<reflink idref="bib8" id="ref28">8</reflink>] established an RT-LAMP assay that allows visual detection by the naked eye. However, the primers were designed based on a single NDV strain's F gene and were not tested against different genotypes. Similarly, Selim et al[<reflink idref="bib11" id="ref29">11</reflink>] developed an RT-LAMP assay specifically for genotype VII NDV, limiting its applicability to that single genotype. More recently, Song et al[<reflink idref="bib12" id="ref30">12</reflink>] developed two sets of RT-LAMP assays targeting the F and HN genes. However, since both genes are less conserved,[<reflink idref="bib3" id="ref31">3</reflink>] the long-term effectiveness of these primers may be reduced due to the ongoing evolution of NDVs.[<reflink idref="bib20" id="ref32">20</reflink>] Furthermore, the HN gene-based primers developed by Song et al[<reflink idref="bib12" id="ref33">12</reflink>] were found to be less effective when used with the RNA extraction-free method established in this study. The LAMP assay established in this study targets the highly conserved L gene, enabling detection of a wide range of NDV genotypes and pathotypes. To date, all published LAMP methods for NDV detection have been based on RNA prepared using conventional RNA extraction kits. Our method eliminates the need for RNA extraction kits, allowing the assay to be performed rapidly and at low cost, making it particularly suitable for field use and in resource-limited settings. The limitation of this assay is its reduced sensitivity in low viral load samples and its evaluation on a limited number of field samples.</p> <hd id="AN0190276331-18">Potential Applications in Surveillance and Outbreak Control</hd> <p>The developed LAMP assay can be employed for routine surveillance of NDV in poultry and for rapid on-site outbreak investigations, particularly in resource-limited settings where conventional laboratory infrastructure is unavailable.</p> <hd id="AN0190276331-19">Conclusions</hd> <p>The LAMP method established in this study offers a rapid, cost-effective, and field-applicable diagnostic tool for the detection of a broad range of NDV genotypes. The RNA extraction-free approach developed in this study enhances the practicality of this method, enabling timely diagnosis and effective response to outbreaks in field and resource-limited settings.</p> <hd id="AN0190276331-20">Ethical Statement</hd> <p>Swab samples were collected from live chickens by a veterinarian in accordance with the World Organization for Animal Health (WOAH) guidelines for the collection and shipment of diagnostic specimens (OIE Terrestrial Manual, 2008) https://<ulink href="http://www.woah.org/fileadmin/Home/eng/Animal%5fHealth%5fin%5fthe%5fWorld/docs/pdf/1.1.01%5fCOLLECTION.pdf">www.woah.org/fileadmin/Home/eng/Animal%5fHealth%5fin%5fthe%5fWorld/docs/pdf/1.1.01%5fCOLLECTION.pdf</ulink>. Informed consent was also obtained from bird owners for the sample collection. Tissue samples used in this study were previously stored at –80°C at the Animal Health Institute in Ethiopia. These samples were originally collected following the same WOAH guidelines for the collection and shipment of diagnostic specimens.</p> <hd id="AN0190276331-21">Acknowledgments</hd> <p>We would like to thank Associate Professor Hiroshi Ito, Laboratory of Veterinary Public Health, Faculty of Agriculture, Tottori University, for kindly providing the NDV isolates of genotype I (Ishii) and genotype IV (Herts) used in this study. We would also like to express our sincere gratitude to Professor Yasuo Inoshima, Joint Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan, for his expert advice during the design of the experiment. The first author (Berihun Dires Mihiretu) would like to express gratitude to Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) for providing a scholarship during the study.</p> <hd id="AN0190276331-22">Author Contributions</hd> <p>All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.</p> <hd id="AN0190276331-23">Disclosure</hd> <p>The authors report no conflicts of interest in this work.</p> <ref id="AN0190276331-24"> <title> REFERENCES </title> <blist> <bibl id="bib1" idref="ref1" type="bt">1</bibl> <bibtext> Yusoff K, Tan WS. Newcastle disease virus: macromolecules and opportunities. Avian Pathol. 2001;30(5):439–455. doi:10.1080/03079450120078626</bibtext> </blist> <blist> <bibl id="bib2" idref="ref2" type="bt">2</bibl> <bibtext> Dimitrov KM, Abolnik C, Afonso CL, et al. Updated unified phylogenetic classification system and revised nomenclature for Newcastle disease virus. Infect Genet Evol. 2019;74:103917. doi:10.1016/j.meegid.2019.103917</bibtext> </blist> <blist> <bibl id="bib3" idref="ref3" type="bt">3</bibl> <bibtext> World Organization for Animal Health (WOAH). Newcastle disease (infection with Newcastle disease virus). 2021.</bibtext> </blist> <blist> <bibl id="bib4" idref="ref4" type="bt">4</bibl> <bibtext> Wise MG, Suarez DL, Seal BS, et al. Development of a real-time reverse-transcription PCR for detection of Newcastle disease virus RNA in clinical samples. J Clin Microbiol. 2004;42(1):329–338. doi:10.1128/JCM.42.1.329-338.2004</bibtext> </blist> <blist> <bibl id="bib5" idref="ref5" type="bt">5</bibl> <bibtext> Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12):e63–e63. doi:10.1093/nar/28.12.e63</bibtext> </blist> <blist> <bibl id="bib6" idref="ref7" type="bt">6</bibl> <bibtext> Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes. 2002;16(3):223–229. doi:10.1006/mcpr.2002.0415</bibtext> </blist> <blist> <bibl id="bib7" idref="ref8" type="bt">7</bibl> <bibtext> Notomi T, Mori Y, Tomita N, Kanda H. Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects. J Microbiol. 2015;53(1):1–5. doi:10.1007/s12275-015-4656-9</bibtext> </blist> <blist> <bibl id="bib8" idref="ref28" type="bt">8</bibl> <bibtext> Kirunda H, Thekisoe O, Kasaija P, et al. Use of reverse transcriptase loop-mediated isothermal amplification assay for field detection of Newcastle disease virus using less invasive samples. Vet World. 2012;5(4):206. doi:10.5455/vetworld.2012.206-212</bibtext> </blist> <blist> <bibl id="bib9" idref="ref25" type="bt">9</bibl> <bibtext> Li Q, Xue C, Qin J, et al. An improved reverse transcription loop-mediated isothermal amplification assay for sensitive and specific detection of Newcastle disease virus. Arch Virol. 2009;154:1433–1440. doi:10.1007/s00705-009-0464-z</bibtext> </blist> <blist> <bibtext> Pham HM, Nakajima C, Ohashi K, Onuma M. Loop-mediated isothermal amplification for rapid detection of Newcastle disease virus. J Clin Microbiol. 2005;43(4):1646–1650. doi:10.1128/JCM.43.4.1646-1650.2005</bibtext> </blist> <blist> <bibtext> Selim K, Adel A, Eid S, Shahein M. Development of real time reverse transcription loop-mediated isothermal amplification assay for rapid detection of genotype VII of Newcastle disease viruses. British Poul Sci. 2022;63(6):864–870. doi:10.1080/00071668.2022.2094219</bibtext> </blist> <blist> <bibtext> Song H-S, Kim H-S, Kim J-Y, Kwon Y-K, Kim H-R. The development of novel reverse transcription loop-mediated isothermal amplification assays for the detection and differentiation of virulent newcastle disease virus. Int J Mol Sci. 2023;24(18):13847. doi:10.3390/ijms241813847</bibtext> </blist> <blist> <bibtext> Barza R, Patel P, Sabatini L, Singh K. Use of a simplified sample processing step without RNA extraction for direct SARS-CoV-2 RT-PCR detection. J Clin Virol. 2020;132:104587. doi:10.1016/j.jcv.2020.104587</bibtext> </blist> <blist> <bibtext> Guan B, Frank KM, Maldonado JO, et al. Sensitive extraction-free SARS-CoV-2 RNA virus detection using a chelating resin. iScience. 2021;24(9):102960. doi:10.1016/j.isci.2021.102960</bibtext> </blist> <blist> <bibtext> Ñique AM, Coronado-Marquina F, Mendez Rico JA, et al. A faster and less costly alternative for RNA extraction of SARS-CoV-2 using proteinase k treatment followed by thermal shock. PLoS One. 2021;16(3):e0248885. doi:10.1371/journal.pone.0248885</bibtext> </blist> <blist> <bibtext> Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021;38(7):3022–3027. doi:10.1093/molbev/msab120</bibtext> </blist> <blist> <bibtext> Clamp M, Cuff J, Searle SM, Barton GJ. The jalview java alignment editor. Bioinformatics. 2004;20(3):426–427. doi:10.1093/bioinformatics/btg430</bibtext> </blist> <blist> <bibtext> Zhang Y, Ren G, Buss J, Barry AJ, Patton GC, Tanner NA. Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride. Biotechniques. 2020;69(3):178–185. doi:10.2144/btn-2020-0078</bibtext> </blist> <blist> <bibtext> Mihiretu BD, Usui T, Chibssa TR, Yamaguchi T. Genetic and antigenic characteristics of genotype VII. 1.1 Newcastle disease viruses currently circulating in Ethiopian chickens. Virol J. 2025;22(1):63. doi:10.1186/s12985-025-02686-x</bibtext> </blist> <blist> <bibtext> Hu Z, He X, Deng J, Hu J, Liu X. Current situation and future direction of Newcastle disease vaccines. Vet Res. 2022;53(1):99. doi:10.1186/s13567-022-01118-w</bibtext> </blist> </ref> <aug> <p>By Berihun Dires Mihiretu; Tatsufumi Usui; Tesfaye Rufael Chibssa; Abde Aliy Mohammed and Tsuyoshi Yamaguchi</p> <p>Reported by Author; Author; Author; Author; Author</p> </aug> <nolink nlid="nl1" bibid="bib12" firstref="ref9"></nolink> <nolink nlid="nl2" bibid="bib16" firstref="ref15"></nolink> <nolink nlid="nl3" bibid="bib17" firstref="ref16"></nolink> <nolink nlid="nl4" bibid="bib14" firstref="ref18"></nolink> <nolink nlid="nl5" bibid="bib18" firstref="ref19"></nolink> <nolink nlid="nl6" bibid="bib19" firstref="ref20"></nolink> <nolink nlid="nl7" bibid="bib10" firstref="ref24"></nolink> <nolink nlid="nl8" bibid="bib11" firstref="ref29"></nolink> <nolink nlid="nl9" bibid="bib20" firstref="ref32"></nolink>
Header DbId: vft
DbLabel: Veterinary Source
An: 190276331
AccessLevel: 3
PubType: Academic Journal
PubTypeId: academicJournal
PreciseRelevancyScore: 0
IllustrationInfo
Items – Name: Title
  Label: Title
  Group: Ti
  Data: Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) for Rapid Diagnosis of Newcastle Disease in Field and Resource-Limited Areas.
– Name: Author
  Label: Authors
  Group: Au
  Data: <searchLink fieldCode="AR" term="%22Mihiretu%2C+Berihun+Dires%22">Mihiretu, Berihun Dires</searchLink> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Usui%2C+Tatsufumi%22">Usui, Tatsufumi</searchLink> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Chibssa%2C+Tesfaye+Rufael%22">Chibssa, Tesfaye Rufael</searchLink> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Mohammed%2C+Abde+Aliy%22">Mohammed, Abde Aliy</searchLink> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Yamaguchi%2C+Tsuyoshi%22">Yamaguchi, Tsuyoshi</searchLink> (AUTHOR)
– Name: TitleSource
  Label: Source
  Group: Src
  Data: <searchLink fieldCode="JN" term="%22Veterinary+Medicine%3A+Research+%26+Reports%22">Veterinary Medicine: Research & Reports</searchLink>. Nov2025, Vol. 16, p39-47. 9p.
– Name: TypeDocument
  Label: Document Type
  Group: TypDoc
  Data: Article
– Name: Subject
  Label: Subjects
  Group: Su
  Data: <searchLink fieldCode="DE" term="%22Newcastle+disease%22">Newcastle disease</searchLink><br /><searchLink fieldCode="DE" term="%22Newcastle+disease+virus%22">Newcastle disease virus</searchLink><br /><searchLink fieldCode="DE" term="%22Diagnosis%22">Diagnosis</searchLink><br /><searchLink fieldCode="DE" term="%22Molecular+diagnosis%22">Molecular diagnosis</searchLink><br /><searchLink fieldCode="DE" term="%22Poultry%22">Poultry</searchLink><br /><searchLink fieldCode="DE" term="%22Loop-mediated+isothermal+amplification%22">Loop-mediated isothermal amplification</searchLink>
– Name: Keyword
  Label: Author-Supplied Keywords
  Group: Kw
  Data: <searchLink fieldCode="DE" term="%22loop+mediated+isothermal+amplification%22">loop mediated isothermal amplification</searchLink><br /><searchLink fieldCode="DE" term="%22RNA+preparation%22">RNA preparation</searchLink>
– Name: Abstract
  Label: Abstract
  Group: Ab
  Data: Background: Newcastle Disease (ND) is a highly contagious and fatal poultry disease caused by Newcastle disease virus (NDV) and has a global distribution. To control this disease, a rapid method for diagnosing it is needed. PCR-based molecular diagnostics such as real-time reverse transcription-PCR (RRT-PCR) for detecting NDV genes have been conducted worldwide. However, because PCR methods are time-consuming and require a well-equipped laboratory, they are not well-suited for use in field and resource limited areas. Methods: In this study, we established a field-friendly loop-mediated isothermal amplification (LAMP) assay for NDV diagnosis using primers targeting the highly conserved L gene, which bypasses the need for RNA extraction and makes it more suitable for field application than previously reported LAMP methods. Results: Our LAMP method is capable of detecting a broad range of NDV genotypes and showed no cross-reactivity with other avian viral diseases or the host genome. The reaction is completed within 35 minutes of incubation at 65°C. NDV was successfully detected directly from swab and tissue samples without the need for conventional RNA extraction. Conclusion: The LAMP method developed in this study offers a rapid, affordable, and field-friendly diagnostic tool for NDV detection. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
  Label:
  Group:
  Data: <i>Copyright of Veterinary Medicine: Research & Reports is the property of Dove Medical Press Ltd and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
– Name: FullTextWordCount
  Label: Full Text Word Count
  Group: FTInfo
  Data: 4246
– Name: ISSN
  Label: ISSN
  Group: ISSN
  Data: 2230-2034
– Name: DOI
  Label: DOI
  Group: ID
  Data: 10.2147/VMRR.S549952
– Name: AN
  Label: Accession Number
  Group: ID
  Data: 190276331
PLink https://erproxy.cvtisr.sk/sfx/access?url=https://search.ebscohost.com/login.aspx?direct=true&site=eds-live&db=vft&AN=190276331
RecordInfo BibRecord:
  BibEntity:
    Identifiers:
      – Type: doi
        Value: 10.2147/VMRR.S549952
    Languages:
      – Code: eng
        Text: English
    PhysicalDescription:
      Pagination:
        PageCount: 9
        StartPage: 39
    Subjects:
      – SubjectFull: Newcastle disease
        Type: general
      – SubjectFull: Newcastle disease virus
        Type: general
      – SubjectFull: Diagnosis
        Type: general
      – SubjectFull: Molecular diagnosis
        Type: general
      – SubjectFull: Poultry
        Type: general
      – SubjectFull: Loop-mediated isothermal amplification
        Type: general
    Titles:
      – TitleFull: Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) for Rapid Diagnosis of Newcastle Disease in Field and Resource-Limited Areas.
        Type: main
  BibRelationships:
    HasContributorRelationships:
      – PersonEntity:
          Name:
            NameFull: Mihiretu, Berihun Dires
      – PersonEntity:
          Name:
            NameFull: Usui, Tatsufumi
      – PersonEntity:
          Name:
            NameFull: Chibssa, Tesfaye Rufael
      – PersonEntity:
          Name:
            NameFull: Mohammed, Abde Aliy
      – PersonEntity:
          Name:
            NameFull: Yamaguchi, Tsuyoshi
    IsPartOfRelationships:
      – BibEntity:
          Dates:
            – D: 01
              M: 11
              Text: Nov2025
              Type: published
              Y: 2025
          Identifiers:
            – Type: issn-print
              Value: 22302034
          Numbering:
            – Type: volume
              Value: 16
          Titles:
            – TitleFull: Veterinary Medicine: Research & Reports
              Type: main
ResultId 1