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Frontiers in Veterinary Science 2024Avian coccidiosis, a parasitic disease prevalent in poultry, is caused by species and leads to significant economic losses. The use of attenuated live oocyst vaccines...
Avian coccidiosis, a parasitic disease prevalent in poultry, is caused by species and leads to significant economic losses. The use of attenuated live oocyst vaccines has been adopted as an alternative to the use of anticoccidial drugs. However, the accurate detection and differentiation of vaccine strains from virulent ones remain challenging. Therefore, this study presents a novel TaqMan polymerase chain reaction (PCR) detection method that offers enhanced sensitivity, specificity, and reproducibility compared with traditional PCR techniques. Through whole-genome resequencing and bioinformatics analysis, we identified a molecular marker gene, Em_marker6, with a unique 21-base pair deletion specific to the attenuated vaccine strain. Optimized primers and probes targeting this marker enabled rapid quantification cycle value achievement and high fluorescence intensity. The standard curve's slope of -3.540 and correlation coefficient of 0.9971 confirmed precise quantification capabilities. The TaqMan PCR method detected as few as 30 plasmid DNA copies and 50 oocysts per reaction, outperforming traditional PCR techniques by an order of magnitude. No cross-reactivity was observed with other wide-type strains or common intestinal pathogens, ensuring the exclusive detection of the EMPY vaccine strain. Weekly testing over 3 weeks demonstrated minimal variability, indicating robust consistency in the method's application. Testing on 61 clinical samples revealed a 57.38% positivity rate for species and 13.11% for the vaccine strain. The Em_marker6 gene exhibited genetic stability across multiple generations, confirming the detection method's robust stability for the attenuated vaccine strain. This study significantly advances the field of avian coccidiosis research and control by providing a valuable tool for monitoring vaccine purity and preventing inadvertent infections in vaccinated flocks, aligning with global efforts to curb antibiotic use in animal feed.
PubMed: 38840634
DOI: 10.3389/fvets.2024.1397166 -
Plant Disease Jun 2024Osmanthus fragrans is an evergreen garden tree species, with high ecological, social, and economic benefits (Lan et al. 2023), which is widely planted in Guizhou...
Osmanthus fragrans is an evergreen garden tree species, with high ecological, social, and economic benefits (Lan et al. 2023), which is widely planted in Guizhou Province. From late April to June 2023, a leaf blight disease was observed on O. fragrans in a bauxite mining area in Qingzhen City, with an incidence of ~50%. Symptoms first appeared at the leaf tip or margin, as irregular brown spots, which gradually coalesced into dark brown patches until the leaves withered and fell off. Symptomatic leaves were collected and surface disinfected with 2% NaClO for 30 s, 75% ethanol for 30 s, rinsed 3 times in sterile ddH2O, air-dried and placed on potato dextrose agar (PDA) medium and incubated at 25°C for 7 d. Fungal colonies on PDA of 9 similar obtained isolates were white, with at least one concentric ring. The reverse was light yellow and gradually turned brown. At 12 d, the pycnidia on PDA was gray to black, spherical or conical, with a diameter of 305.15 μm (n=20). The conidial horns oozed out from pycnidia after 25 d of incubation on Pinus massoniana needles. The alpha conidia were unicellular, fusiform, hyaline, had a guttule at each end, and measured 6.24 ± 0.10 μm × 2.48 ± 0.04 μm (n=50). No beta or gamma conidia were observed. The morphological characteristics were likely to Diaporthe spp. (Gomes et al. 2013). DNA of isolates GH02, GH06 and GH08 was extracted. The internal transcribed spacer region (ITS) and partial sequences of translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), beta-tubulin (TUB2), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R, CAL228F/CAL737R (Carbone and Kohn, 1999), βt2a/βt2b and CYLH3F/H3-1b (Crous et al. 2004; Glass and Donaldson, 1995), respectively. The sequences of ITS, TEF-1α, TUB2, CAL and HIS were deposited in GenBank (GH02: PP813499, PP813844, PP813846, PP813848 and PP813850; GH06: PP813500, PP813845, PP813847, PP813849 and PP813851; GH08: PP507168 and PP529956 to PP529959). BLAST results showed the sequences of GH08 were highly identical to sequences of Phomopsis mahothocarpi (NR147522 [ITS], 527/530), P. mahothocarpi (MW700277 [TEF-1α], 367/372), D. eres (OR885862 [TUB], 513/513), D. celeris (ON221721 [CAL], 484/486), and D. eres (OP968956 [HIS], 477/477). A phylogenetic tree constructed with MEGA X using Neighbor-Joining algorithm (Felsenstein, 1985) indicated the isolate GH02, GH06 and GH08 separated from D. eres CBS 297.77 previously reported from O. aquifolium in Netherlands, as well as D. osmanthi and D. fusicola from O. fragrans in China (Gomes et al. 2013; Long et al. 2019; Si et al. 2021). Based on these results, the three isolates were identified as D. eres (Chaisiri et al., 2021). The isolate GH08 was deposited in the Forest Protection Laboratory, Guizhou University. To confirm pathogenicity, spore suspensions (1×10 spores/mL) of GH08 were sprayed on healthy detached leaves (n=10) and leaves of 3-year-old potted O. fragrans seedlings (n=8). An equal volume of sterile water was sprayed for the control. Then they were placed at 20°C and 70-80% RH. Similar leaf blight symptoms appeared after 5 and 15d on the inoculated leaves and seedlings, respectively. The re-isolated fungus, was identical to D. eres based on morphological and molecular analysis, thus fulfilling Koch's postulates. To our knowledge, this is the first report of D. eres causing leaf blight of O. fragrans in China, supporting a basis for developing effective methods to manage this disease.
PubMed: 38840487
DOI: 10.1094/PDIS-04-24-0765-PDN -
Plant Disease Jun 2024Hydrangea (Hydrangea macrophylla), commonly referred to as big leaf hydrangea, is a species within the Hydrangeaceae family notable for its ornamental value....
Hydrangea (Hydrangea macrophylla), commonly referred to as big leaf hydrangea, is a species within the Hydrangeaceae family notable for its ornamental value. Characterized by its vividly colored sepals and lush, striking inflorescences, this species is globally esteemed as both a potted and landscape plant. Notably, in 2022, an alarming incidence of stem rot was observed in approximately 40% of H. macrophylla plants aged between six and twelve months within 16 greenhouses situated in Nanjing City (N 31°14', E 118°22'), Jiangsu Province, China. Initial symptoms of the disease manifested as wet gray-black spots at the base of the seedlings and stems, progressing to a necrotic gray-white discoloration in the stems and accompanied by the growth of gray mold on the affected parts. This infection ultimately led to the wilting of the leaves and the death of the seedlings. For pathogen identification, stem tissues at the interface of diseased and healthy sections were excised, surface-sterilized with 75 % ethanol for 30 s, followed by a 2 - 3 min treatment with 3% sodium hypochlorite, and subsequently rinsed three times with sterile water before air drying. Sections measuring 2 - 3 mm were then cultured on potato dextrose agar (PDA) medium, supplemented with 50 mg/mL rifampicin (RFP), and incubated at 25 ℃ for 3 - 5 d (Zhou et al. 2022). Upon 2 - 3 days of incubation, notable growth of fungal colonies was observed. Mycelial clusters from the periphery of these colonies were subsequently transferred to fresh PDA plates and incubated at 25 ℃ for an additional 5 - 7 d. A particular colony, designated JSNJ2022-2 and now preserved at the Jiangsu Academy of Agricultural Sciences, was selected for detailed examination. This colony exhibited a flocculent texture, with a coloration ranging from grey-white to light brown. It was characterized by the presence of irregularly formed, hard sclerotia within the hyphae. The conidiophores were observed to be slender and erect, featuring dendritic branches at their extremities. The conidia were clustered on the conidiophore like grapes. These conidia were generally colorless or grey, oval in shape, smooth and transparent, and measured between 6.4 - 12.2 × 7.3 - 18.2 μm (n = 50). For genetic analysis, genomic DNA (gDNA) was extracted using the DNA secure Plant Kit (Tiangen Biotech, Beijing, China). Polymerase chain reaction (PCR) amplification was performed using a set of universal primers of ITS1/ITS4 (White et al. 1990), primers corresponding to the specific sequences of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) (Yang et al. 2020). The resultant PCR products were sequenced, and the resulting sequences were submitted to the GenBank database, under the accession numbers OP131597, OP142320, OP142321, and OP142322, respectively. BLAST analysis of the sequences obtained from the isolate JSNJ2022-2 revealed a high degree of genetic similarity, ranging from 99 to 100%, with known sequences of Botrytis cinerea (accessions MK051124.1, MH796662.1, MH479931.1, and KU760986.1). To elucidate the phylogenetic position of the isolate, a phylogenetic tree was constructed using the maximum likelihood method, supported by 1,000 bootstrap replications, in the Mega7 software (Kumar et al. 2016). The results of this analysis confirmed that the strains under study clustered within the same branch as B. cinerea. To establish the pathogenicity of the isolate, Koch's postulates (Falkow 1988) were employed. Healthy potted H. macrophylla seedlings, approximately three months old, were wound inoculated at the base of the seedlings with a 6 mm diameter mycelium plug of JSNJ2002-2 cultivated on PDA for 3 days, which was subsequently covered with moistened degreasing cotton. Control plants were treated with moistened degreasing cloths minus the pathogen. Post-inoculation, these plants were placed in a growth chamber maintained at 25 ℃ with a relative humidity range of 60 - 80%. After a 3-d incubation period, the inoculated plants displayed symptoms identical to those initially observed in the greenhouse. The pathogen was successfully re-isolated from these inoculated plants and was morphologically re-confirmed as B. cinerea, thus satisfying the criteria of Koch's postulates. To our knowledge, this report represents the first documented incidence of B. cinerea causing stem rot in H. macrophylla in China.
PubMed: 38840485
DOI: 10.1094/PDIS-01-24-0230-PDN -
Tropical Medicine and Health Jun 2024This study aimed to develop a multiplex PCR assay for simultaneous detection of major Gram-negative etiologies of septicemia and evaluate its performance.
AIM
This study aimed to develop a multiplex PCR assay for simultaneous detection of major Gram-negative etiologies of septicemia and evaluate its performance.
METHODS
Multiplex PCR (mPCR) assays were developed targeting 11 bacterial strains. Species-specific primers were confirmed using known clinical isolates and standard strains. Gradient PCR was performed on each primer against its target bacterial gene to determine its optimal amplification condition. The minimum detectable DNA concentration of the two assays was evaluated by adjusting bacterial DNA concentration to 100 ng/μL and, tenfold serially diluting it up to 10 pg/μL with DNAse-free water. The diagnostic accuracy of mPCR assays was established by subjecting the assays to 60 clinical blood samples.
RESULTS
Two mPCR assays were developed. Optimal primer annealing temperature of 55 °C was established and utilized in the final amplification conditions. The assays detected all targeted bacteria, with a 100 pg minimum detectable DNA concentration. Pathogens were not detected directly from whole blood, but after 4 h and 8 h of incubation, 41% (5/12) and 100% (12/12) of the bacteria were detected in culture fluids, respectively. The assays also identified Salmonella spp. and Klebsiella pneumoniae co-infections and extra pathogens (1 E. coli and 2 K. pneumoniae) compared with culture. The sensitivity and specificity of the mPCR were 100.0% (71.7-100.0) and 98.0% (90.7-99.0), respectively. The area under the ROC curve was 1.00 (1.00-1.00).
CONCLUSIONS
The mPCR assays demonstrated substantial potential as a rapid tool for septicemia diagnosis alongside the traditional blood culture method. Notably, it was able to identify additional isolates, detect co-infections, and efficiently detect low bacterial DNA loads with high sensitivity, implying its value in enhancing efficiency of diagnosis of septicemia.
PubMed: 38840209
DOI: 10.1186/s41182-024-00606-3 -
Veterinary Journal (London, England :... Jun 2024Penile squamous cell carcinomas (SCCs) are common, potentially life-threatening neoplasms of horses. They are well-recognized to be caused by Equus caballus...
Penile squamous cell carcinomas (SCCs) are common, potentially life-threatening neoplasms of horses. They are well-recognized to be caused by Equus caballus papillomavirus (EcPV) type 2, although EcPV2 cannot be detected in all cases. A 23-year-old standardbred gelding developed multiple penile in situ and invasive SCCs that contained histological evidence of PV infection. By using both consensus and specific PCR primers, these lesions were found to contain EcPV7 DNA, but not DNA from EcPV2 or any other PV type. To determine how frequently EcPV7 is present in equine penile SCCs, specific primers were used to detect EcPV2 and EcPV7 in a series of 20 archived samples. EcPV7 was the only PV detected in one, both EcPV2 and 7 were detected in five, and only EcPV2 was detected in 14 SCCs. EcPV7 DNA was also detected in three of 10 archived oropharyngeal SCCs, although only as a co- infection with EcPV2. This is the first report of EcPV7 causing disease in horses. These results suggest EcPV7 could cause a subset of equine penile SCCs, and this is the first evidence that PV types other than EcPV2 can cause these neoplasms. The detection of EcPV7 in the oropharyngeal SCCs suggests a potential role of this PV type in the development of these SCCs. There were no clinical or histological features that differentiated lesions containing EcPV7 DNA from those containing EcPV2 DNA. If EcPV7 causes a proportion of equine penile SCCs, vaccines to prevent EcPV2 infection may not prevent all equine penile SCCs.
PubMed: 38838769
DOI: 10.1016/j.tvjl.2024.106155 -
PloS One 2024Areca palm velarivirus 1 (APV1) is one of the main pathogen causing yellow leaf disease, and leading to considerable losses in the Areca palm industry. The detection...
Areca palm velarivirus 1 (APV1) is one of the main pathogen causing yellow leaf disease, and leading to considerable losses in the Areca palm industry. The detection methods for APV1 are primarily based on phenotype determination and molecular techniques, such as polymerase chain reaction (PCR). However, a single PCR has limitations in accuracy and sensitivity. Therefore, in the present study, we established a dual RT-PCR APV1-detection system with enhanced accuracy and sensitivity using two pairs of specific primers, YLDV2-F/YLDV2-R and YLDV4-F/YLDV4-R. Moreover, two cDNA fragments covering different regions of the viral genome were simultaneously amplified, with PCR amplicon of 311 and 499 bp, respectively. The dual RT-PCR detection system successfully amplified the two target regions of the APV1, demonstrating high specificity and sensitivity and compensating for the limitations of single-primer detection methods. We tested 60 Areca palm samples from different geographical regions, highlighting its advantages in that the dual RT-PCR system efficiently and accurately detected APV1 in samples across diverse areas. The dual RT-PCR APV1 detection system provides a rapid, accurate, and sensitive method for detecting the virus and offers valuable technical support for research in preventing and managing yellow leaf diseases caused by APV1 in Areca palms. Moreover, the findings of this study can serve as a reference for establishing similar plants viral detection systems in the future.
Topics: Reverse Transcriptase Polymerase Chain Reaction; Plant Diseases; Arecaceae; Sensitivity and Specificity; DNA Primers; RNA, Viral
PubMed: 38838001
DOI: 10.1371/journal.pone.0303941 -
Plant Disease Jun 2024Pumpkin (Cucurbita moschata), which belongs to the gourd family (Cucurbitaceae), is widely planted throughout the world. In June 2023, many pumpkin plants (cv. Miben)...
Pumpkin (Cucurbita moschata), which belongs to the gourd family (Cucurbitaceae), is widely planted throughout the world. In June 2023, many pumpkin plants (cv. Miben) displayed leaf blight and chlorosis in fields located in Suizhou (31.99°N, 113.02°E), Hubei Province, China. The disease incidence ranged from 30 to 40% in nine fields, 6.3 ha in total. The symptoms were irregularly shaped lesions that expanded along the mid-vein until the leaf turned brown and wilted. Fungal isolations were performed as described previously (Liu et al. 2023). Twenty pumpkin leaf samples with typical symptoms were collected and cut into 1 cm×1 cm pieces. The diseased tissue was surface-sterilized in 75% ethanol for 30 sec, plated on potato dextrose agar (PDA) medium and incubated at 25℃ for 3 days. Then, the emerging single fungal hyphal tip was transferred onto PDA plates to obtain purified isolates. A total of eighteen isolates on PDA plates were initially white and then developed to dark gray. The 5-day-old cultures growing on mung bean medium produced conidia that were black, single-celled, smooth, spherical or oblate, and ranged in size from 14.5 to 20.8 μm×13.3 to 20.5 μm (n=50). Therefore, the isolates were morphologically identified as Nigrospora sphaerica. Moreover, the genomic DNA of the isolates (HB-P1,HB-P2, and HB-P3) was extracted for amplification and sequencing of the regions of internal transcribed spacer (ITS) (White et al. 1990), nuclear large subunit rRNA (nLSU) (O'Donnell 1992; Rehner and Samuels 1994), and β-tubulin (TUB2) (Glass and Donaldson 1995), with primers ITS1/ITS4, LROR/LR3, and Bt2a/Bt2b, respectively. Sequences were submitted to GenBank under accession numbers PP348112, PP348113, PP348114 (ITS), PP411414, PP411415, PP411416 (nLSU), and PP357438, PP357439, PP357440 (TUB2). BLASTn showed that the sequences ITS, nLSU, and TUB2 of HB-P1, HB-P2, and HB-P3 had >99% nucleotide identities ((ITS: 100%, 508/508 bp, MF996488.1; 99.8%, 506/507, ON326588.1; 100%, 500/500 ,MK748317.1), (nLSU: 99.83%, 573/574, KT462720.1; 99.83% , 574/575 bp, KT462720.1; 99.65%, 575/577, KT462720.1), and (TUB2: 100%, 388/388, MN719407.1; 99.74%, 387/388, MN719407.1; 100%, 387/387, MN719407.1)) with Nigrospora sphaerica, respectively. A multilocus (ITS, nLSU and TUB2) phylogenetic analysis indicated that the isolates were Nigrospora sphaerica. Pathogenicity of three isolates were tested on pumpkin plants (cv. Miben). Fifteen pumpkin plants were inoculated by spraying the leaves (1×106 spores/ml), respectively, and 10 pumpkin plants were treated with sterile water as a negative control. All plants were incubated in an artificial climate box (LongYue, ShangHai) at 25℃ for 12 days. The experiment was repeated three times. Twelve days later, the inoculated pumpkin plants developed symptoms of leaf blight, while the control plants remained healthy. Then, pathogens were re-isolated from the each leaf of inoculated pumpkin plants and not from the control plants. Nigrospora sphaerica has been previously reported to cause leaf spot on watermelon in Malaysia (Ismail and Abd Razak 2021). To our knowledge, this is the first report of N. sphaerica causing leaf blight on pumpkin in China. This new disease can cause leaf blight, which may affect pumpkin productivity.
PubMed: 38831590
DOI: 10.1094/PDIS-03-24-0571-PDN -
Plant Disease Jun 2024Maxim. (), one of the Chinese herbal medicines, is an economically important crop in Anhui Province, China. In recent years, gummy stem blight disease, a major disease...
Maxim. (), one of the Chinese herbal medicines, is an economically important crop in Anhui Province, China. In recent years, gummy stem blight disease, a major disease of cucurbits, was widespread in many plantations. The initial symptoms on the naturally infected stems appeared as dark brown water-soaked lesions, and as the disease progressed, vines of gradually withered. On leaves, brown water-soaked lesions were visible initially, and then lesions enlarged and coalesced, resulting in extensive necrosis of leaves. On fruit, lesions covered with the white mycelium were nearly circular and tan to brown initially. Subsequently, the diseased fruit turned black and rotten commonly known as fruit rot or black rot. A -like organism was consistently isolated from symptomatic stems, leaves and fruits. Fungal isolates were initially white and later turned dark grey or black with woolly to floccose aerial mycelium on PDA medium. Twenty-four isolates from different plantations were selected for further morphological studies. Pycnidia and conidia were formed after inoculating on cucumber fruit for 3 days. Pycnidia were globose to sub-globose, brown, ostiolate and 106.7 to 213.6 μm (average 160.1 μm, n = 50) in diameter. Conidia were hyaline, ellipsoidal, aseptate or one-septate, slightly constricted at the septa, 6.1 to 13.6 × 3.5 to 4.8 μm (average 9.9 × 4.1 μm, n = 50), and contained two or more oil drops. Three different loci of the genomic DNA, including the nuclear ribosome DNA internal transcribed spacer (), RNA polymerase II second-largest subunit (), and β-tubulin () genes., were amplified using primers ITS1/ITS4 (White et al. 1990), RBP2DF/RBP2DR (Lawrence et al. 2013), and T1/β-Sandy-R (O' Donnell and Cigelnik 1997; Stukenbrock et al. 2012), respectively and sequenced. A phylogenetic tree was built based on analysis of , , and sequences that deposited in GenBank (MW485497-MW485502 for ITS, MW531661-MW531666 for RPB2, and MW531667-MW531672 for TUB2), using the maximum likelihood method. The phylogenetic tree showed that the isolates fell into a single clade with . On the basis of morphological and molecular characteristics, the isolates obtained from were identified as . Pathogenicity tests were carried out on stems and leaves of 4-week-old seedlings and on immature fruit collected from adult plants. The epidermis, previously injured with a syringe needle, was inoculated with 5-mm-diameter mycelial plugs, and the inoculated areas were then wrapped in water-soaked cotton. Controls were similarly inoculated with agar plugs. The diameters of lesions were measured in two perpendicular directions. Re-isolations from the stem and leaf lesions were performed on the PDA medium. , was re-identified based on its colony and conidial characteristics and, therefore, completed Koch's postulates. Gummy stem blight caused by has been reported in a wide range of hosts, including cucumber, luffa, pumpkin, gourd, muskmelon, cantaloupe, and watermelon (Jiang et al. 2015; Keinath 2011; Zhao et al. 2019). To our knowledge, this is the first report of gummy Stem blight disease on caused by in China. The research provides a basis for the development and implementation of effective management strategies. Pathogenicity tests were carried out on stems and leaves of 4-week-old seedlings and on immature fruits collected from adult plants. The epidermis, previously injured with a syringe needle, was inoculated with 5-mm-diameter mycelial plugs, and the inoculated areas were then wrapped in water-soaked cotton. Controls were treated similarly but inoculated with agar plugs. Diameters of lesions were measured in two mutually perpendicular directions. Reisolations from the lesions were performed on PDA medium, and was re-identified based on its colony and conidial characteristics to complete Koch's postulates. Gummy stem blight caused by have been reported in a wide range of hosts, including cucumber, luffa, pumpkin, gourd, muskmelon, cantaloupe, and watermelon (Jiang et al. 2015; Keinath 2011; Zhao et al. 2019). To our knowledge, this is the first report of gummy Stem blight disease on caused by in China. The research provides a basis for the development and implementation of effective management strategies.
PubMed: 38831589
DOI: 10.1094/PDIS-09-23-1782-PDN -
BMC Veterinary Research May 2024This study investigates the genetic characteristics of Capillaria isolates from the infected fish, Bagrus bajad, and their relation to human Capillaria philippinensis...
BACKGROUND
This study investigates the genetic characteristics of Capillaria isolates from the infected fish, Bagrus bajad, and their relation to human Capillaria philippinensis using Random Amplified Polymorphic DNA (RAPD-PCR) analysis. Fifteen fish Capillaria were isolated and compared to identified human C. philippinensis using six primers: M-are, M-1, G-7, G-11, G-15, and G-18.
RESULTS
All six primers successfully amplified DNA, highlighting their efficacy in distinguishing between human and fish Capillaria isolates. The analysis revealed distinctive banding patterns between fish and human isolates, with variations in size and number of DNA fragments. Additionally, genetic similarity analysis showed intriguing patterns of relatedness, with certain pairs exhibiting high similarity percentages. Comparative assessment of RAPD polymorphism demonstrated consistent findings of 100% polymorphism across all primers. The Unweighted Pair Group Method with Arithmetic Mean Algorithm (UPGMA) evaluated the closest relationship between human and fish isolates. These results underscore the utility of RAPD analysis in delineating the genetic diversity among Capillaria isolates from different hosts.
CONCLUSION
Overall, this study contributes to our understanding of the genetic variability and relatedness among Capillaria isolates, shedding light on their evolutionary dynamics and zoonotic potential.
Topics: Animals; Fish Diseases; Egypt; Random Amplified Polymorphic DNA Technique; Genetic Variation; Capillaria; Enoplida Infections; Phylogeny; Humans
PubMed: 38822316
DOI: 10.1186/s12917-024-04076-x -
BMC Ecology and Evolution May 2024Monitoring mollusk biodiversity is a great challenge due to their large diversity and broad distribution. Environmental DNA (eDNA) technology is increasingly applied for... (Comparative Study)
Comparative Study
Monitoring mollusk biodiversity is a great challenge due to their large diversity and broad distribution. Environmental DNA (eDNA) technology is increasingly applied for biodiversity monitoring, but relevant studies on marine mollusks are still limited. Although previous studies have developed several pairs of primers for mollusk eDNA analyses, most of them targeted only a small group of mollusks. In this study, seven primers were designed for the mollusk community and validated and compared with eight pairs of published primers to select the best candidates. After in silico test, MollCOI154 and MollCOI255 primers showed non-specific amplification, and same results were also obtained in published primers (COI204, Sepi, and veneroida). Moll12S100, Moll12S195 and Moll16S primers failed to amplify across all genomic DNA from selected mollusk. Except Moll16S, all developed and two published (unionoida and veneroida) primers were successfully amplified on four eDNA samples from Yangtze River estuary. After annotation of the amplified sequences, MollCOI253 showed higher annotation of the amplification results than the other primers. In conclusion, MollCOI253 had better performance in terms of amplification success and specificity, and can provide technical support for eDNA-based research, which will be beneficial for molluscan biodiversity investigation and conservation.
Topics: Mollusca; Animals; DNA Barcoding, Taxonomic; DNA, Environmental; DNA Primers; Biodiversity
PubMed: 38822255
DOI: 10.1186/s12862-024-02265-8