Species-specific Real-time PCR Assay Research Articles

Development of Single-Nucleotide Polymorphism (SNP)-Based Species-Specific Real-Time PCR Assays for Authenticating Five Highly Priced Tuna.

Tuna are economically important as food resources in food markets. However, because tuna is often processed into steaks or fillets, the meat can be difficult to identify through morphological features. For effective fishery management and to protect the rights of consumers, it is necessary to develop a molecular method to accurately identify the species used in tuna products. Herein, we discovered five single-nucleotide polymorphism (SNP) sites via 2b-RAD sequencing and developed five SNP-based real-time polymerase chain reaction assays for the rapid identification of five highly priced tuna species. Three species-specific TaqMan systems were designed to identify albacore tuna (Thunnus alalunga), bigeye tuna (T. obesus), and southern bluefin tuna (T. maccoyii) and two cycling systems were designed to identify yellowfin tuna (T. albacares) and Atlantic bluefin tuna (T. thynnus). The systems showed good specificity and sensitivity (sensitivity of 0.0002 ng μL-1 for albacore tuna, bigeye tuna, and southern bluefin tuna and 0.002 ng μL-1 for yellowfin tuna and Atlantic bluefin tuna). Both systems were able to distinguish the target species from other species in a specific, sensitive, and accurate manner. Thus, these methods can be employed for the identification of species used in tuna products, protecting consumers and producers from economic fraud.

New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple.

Bitter rot of apple is an economically important worldwide disease caused by different Colletotrichum species, depending on many factors such as climate, geography, other hosts, and crop management practices. Culture, morphology, and single-locus sequencing-based methods for identifying the Colletotrichum species are severely limited in effectiveness, while the multilocus sequence typing methods available for delineating species are costly, time-intensive, and require high expertise. We developed species-specific hydrolysis probe real-time PCR assays for the following nine Colletotrichum species causing bitter rot in the Mid-Atlantic U.S.A.: C. fructicola, C. chrysophilum, C. noveboracense, C. gloeosporioides s.s., C. henanense, C. siamense and C. theobromicola from the C. gloeosporioides species complex, and C. fioriniae and C. nymphaeae from the C. acutatum species complex. After searching 14 gene regions, we designed primers and probes in 5 of them for the nine target species. Four primer-probe set pairs were able to be duplexed. Sensitivity tests showed as little as 0.5 pg DNA were detectable. These real-time PCR assays will provide rapid and reliable identification of these key Colletotrichum species and will be critically important for studies aiming to elucidate their biology, epidemiology, and management on apples as the number one produced and consumed tree fruit in the U.S.A.

Whole-genome long-read sequencing to unveil Enterococcus antimicrobial resistance in dairy cattle farms exposed a widespread occurrence of Enterococcus lactis.

Enterococcus faecalis (Efs) and Enterococcus faecium (Efm) are major causes of multiresistant healthcare-associated or nosocomial infections. Efm has been traditionally divided into clades A (healthcare associated) and B (community associated) but clade B has been recently reassigned to Enterococcus lactis (Elc). However, identification techniques do not routinely differentiate Elc from Efm. As part of a longitudinal study to investigate the antimicrobial resistance of Enterococcus in dairy cattle, isolates initially identified as Efm were confirmed as Elc after Oxford-Nanopore long-fragment whole-genome sequencing and genome comparisons. An Efm-specific PCR assay was developed and used to identify isolates recovered from animal feces on five farms, resulting in 44 Efs, 23 Efm, and 59 Elc. Resistance, determined by broth microdilution, was more frequent in Efs than in Efm and Elc but all isolates were susceptible to ampicillin, daptomycin, teicoplanin, tigecycline, and vancomycin. Genome sequencing analysis of 32 isolates identified 23 antimicrobial resistance genes (ARGs, mostly plasmid-located) and 2 single nucleotide polymorphisms associated with resistance to 10 antimicrobial classes, showing high concordance with phenotypic resistance. Notably, linezolid resistance in Efm was encoded by the optrA gene, located in plasmids downstream of the fexA gene. Although most Elc lacked virulence factors and genetic determinants of resistance, one isolate carried a plasmid with eight ARGs. This study showed that Elc is more prevalent than Efm in dairy cattle but carries fewer ARGs and virulence genes. However, Elc can carry multi-drug-resistant plasmids like those harbored by Efm and could act as a donor of ARGs for other pathogenic enterococcal species.IMPORTANCEEnterococcus species identification is crucial due to differences in pathogenicity and antibiotic resistance profiles. The failure of traditional methods or whole-genome sequencing-based taxonomic classifiers to distinguish Enterococcus lactis (Elc) from Enterococcus faecium (Efm) results in a biased interpretation of Efm epidemiology. The Efm species-specific real-time PCR assay developed here will help to properly identify Efm (only the formerly known clade A) in future studies. Here, we showed that Elc is prevalent in dairy cattle, and although this species carries fewer genetic determinants of resistance (GDRs) than Enterococcus faecalis (Efs) and Efm, it can carry multi-drug-resistant (MDR) plasmids and could act as a donor of resistance genes for other pathogenic enterococcal species. Although all isolates (Efs, Efm, and Elc) were susceptible to critically or highly important antibiotics like daptomycin, teicoplanin, tigecycline, and vancomycin, the presence of GDRs in MDR-plasmids is a concern since antimicrobials commonly used in livestock could co-select and confer resistance to critically important antimicrobials not used in food-producing animals.

High-Sensitivity ITS Real-Time PCR Assays for Detection of Ceratocystis lukuohia and Ceratocystis huliohia in Soil and Air Samples

Rapid Ohia Death (ROD) is caused by two pathogens, Ceratocystis lukuohia and Ceratocystis huliohia. The established species-specific real-time PCR assays targeting the single-copy cerato-platanin gene require DNA from between 2 and 16 spores per reaction for consistent detection, which is suitable for analysis of infected plant tissue but not sensitive enough to consistently detect low spore loads in environmental samples. Here, we present two redesigned qPCR assays targeting the first internal transcribed spacer (ITS) region of the multi-copy ribosomal DNA operon from the respective species, both capable of consistent detection of the pathogen at concentrations as low as 1 fg per reaction, less than the size of the haploid genome. Due to this increased sensitivity, these ITS qPCR assays are superior for analysis of DNA extracted from material collected in airborne particle samplers and from soil. Here, we demonstrate the utility of these qPCR assays for the characterization of windblown and soilborne dispersal of the pathogens, which is critical for the development of management practices that mitigate disease spread. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Occurrence of canine subcutaneous dirofilariosis (Dirofilaria repens) in Switzerland

Infectious diseases are increasingly introduced into Switzerland due to the increased travel activity in recent decades and the import of dogs. Dirofilariasis, caused by Dirofilaria immitis or D. repens, is one of them. An infection with D. repens, the cause of canine subcutaneous dirofilariosis, is often asymptomatic in dogs, but represents a potential zoonotic disease risk for humans. Due to the rapidly increasing number of human cases, D. repens is considered an emerging zoonosis in north-eastern Europe. The prevalence of D. repens infections in dogs and humans in Switzerland is unknown. Since 2016 the analysing diagnostic laboratory provided with the newly introduced filaria PCR a realiable diagnostic test to differentiate betweeen D. immitis and D. repens. Total nucleic acid (DNA and RNA) was extracted from 200 μl EDTA blood without prior enrichment followed by a species-specific real-time PCR assay. The analyses for Dirofilariae in the years 2016 to 2021 were examined in a descriptive retrospective study and the proportion of positive tests per year (prevalence with 95 % confidence interval) was calculated. Furthermore, blood samples of 50 imported dogs to Switzerland were analyzed in an exploratory cross-sectional study for the presence of dirofilaria. No D. repens positive case was found in the first two years after the introduction of the PCR. In 2018 five of 546 analyzed samples (5/546, 0,9 %, 95 % confidence interval [95 % CI]=0,7 - 1,2 %) tested positive for D. repens, in 2019 four of 591 (0,7 %, 95 % CI=0,5 - 0,9 %), in 2020 15 of 783 (1,9 %, 95 % CI=1,6 - 2,3 %) and in 2021 eleven of 1058 samples (1,0 %, 95 % CI=0,8 - 1,3 %). In the exploratory cross-sectional study, four of the 50 examined dogs were positive for D. repens (8 %, 95 % CI=2,6 - 20,1 %). One dog had a concurrent infection with D. immitis and D. repens. All four positive tested dogs were imported from Hungary. Potentially zoonotic infections with D. repens occur in dogs living in Switzerland. This disease should be included in the differential diagnoses of imported dogs and a monitoring during routine health checks should be done more often. The veterinary profession can thus assume its responsibility for the prevention of zoonoses as part of a One Health approach.

Real-time PCR assay for detection of Environmental DNA from <i>Chironex yamaguchii</i>

The box jellyfish Chironex yamaguchii is a highly venomous cubomedusa, also known as “habu-kurage” in Japan. It is a significant threat to marine activities in Okinawa in southern Japan. Annually, envenomation of the species causes nearly 100 to 200 injuries in the Okinawa Prefecture. The present study developed a species-specific real-time PCR assay to analyze environmental DNA (eDNA) for rapid detection of jellyfish in water samples. Primers and probe were designed based on the sequences of the mitochondrial cytochrome c oxidase subunit I region using tissue samples of nine specimens obtained from the Okinawa Prefecture. The specificity of the real-time PCR assay was confirmed by amplifying the DNA samples of 10 different box jellyfish species. A practical assay for eDNA detection was successful on water samples obtained from the natural habitat of C. yamaguchii. In addition, the degradation rate of eDNA in the tank experiment was 65% of the initial concentration at 1 h, 22% at 12 h, and 3% at 48 h.

Molecular detection of Escherichia coli isolated from human, animal and environmental samples targeting mdh gene: A simple one health approach

Background: Escherichia coli cause a wide range of infections in humans and animals in hospitals and community associated with multidrug-resistance, and serves as bioindicator of fecal contamination as well as AMR. Aim: Early detection of this bioindicator from humans, animals and environment facilitates characterization with respect to AMR and bacterial forensics for effective public health and infection control measurements. Method: We investigated a simple and rapid species-specific end-point and real-time PCR assays targeting malate dehydrogenase (mdh) gene to detect Escherichia coli isolated from livestock, human, and environmental water in Midnapore, West Bengal, India in a timely and cost-effective approach. Results: Of 126 E. coli isolates, 53, 41 and 32 were cultured from livestock, human and environmental water. The mdh gene was present in 120 (95.2%) isolates, including all environmental isolates. Two (3.8%) animal and four human (9.8%) isolates were discordant between culture and PCR results. The PCR assays produced results in about 90 min with a running cost of US$3-5 per sample. Conclusion: Both the PCR assays may be utilized in detection of Escherichia coli depending on the laboratory infrastructure.

First Molecular Detection of Pathogens Leptospira in Common Rodent Captured in North Algeria Urban Areas.

Leptospirosis is an anthropozoonosis disease of worldwide distribution caused by mobile spirochetes of the genus Leptospira and rodents, mainly rats, are described as its primary reservoir. In Algeria, there is limited data about the prevalence of Leptospira spp. in humans and animals, as well as Leptospira carriage in wild rodents. The study aimed to highlight the importance of rodents as a reservoir of Leptospira bacterium in Blida city in Algeria by detecting and identifying circulating Leptospira species in the rodent population. A total of 101 rodents, 95 Rattus Norvegicus, 5 Rattus Rattus, and 1 Mus Musculus were captured and tested for pathogenic Leptospira spp. byreal-time PCR targeting the Leptospira 16S rRNA (rrs) gene, revealing a total prevalence of 40.6%, 95% IC [30.9-50.8%]. Positive samples were subjected to species-specific real-time PCR assays targeting L. interrogans, L. noguchii, L. borgpetersenii, and L. kirschneri for species identification. However, positive samples for which Leptospira-species could not be determined were subjected to conventional PCR targeting the partial 16S rRNA (rrs) gene, and amplified DNA was subjected to sequencing. Leptospira spp. was detected in 36 kidney, 16 urine, and three lung specimens. L. interrogans was identified in 39 rodents and L. borpetersenii in one rodent; however, one rodent with renal carriage could not be typed due to poor DNA quality. This study provides the first description of pathogenic Leptospira spp. in wild rodents in Algeria. These findings suggest a high potential risk of leptospirosis transmission from rodents to humans and animals in Algeria and therefore imply the adoption of prophylactic measures. In addition, further studies, including different animals and rodent species, should be conducted to clarify the epidemiology of this disease in Algeria.

Molecular survey of Babesia parasites in Kenya: first detailed report on occurrence of Babesia bovis in cattle

BackgroundAmong protozoan parasites in the genus Babesia, Babesia bigemina is endemic and widespread in the East African region while the status of the more pathogenic Babesia bovis remains unclear despite the presence of the tick vector, Rhipicephalus microplus, which transmits both species. Recent studies have confirmed the occurrence of R. microplus in coastal Kenya, and although B. bovis DNA has previously been detected in cattle blood in Kenya, no surveillance has been done to establish its prevalence. This study therefore investigated the occurrence of B. bovis in cattle in Kwale County, Kenya, where R. microplus is present in large numbers.MethodsA species-specific multiplex TaqMan real-time PCR assay targeting two Babesia bovis genes, 18S ribosomal RNA and mitochondrially-encoded cytochrome b and B. bigemina cytochrome b gene was used to screen 506 cattle blood DNA samples collected from Kwale County for presence of Babesia parasite DNA. A sub-set of 29 B. bovis real-time PCR-positive samples were further amplified using a B. bovis-specific spherical body protein-4 (SBP-4) nested PCR and the resulting products sequenced to confirm the presence of B. bovis.ResultsA total of 131 animals (25.8%) were found to have bovine babesiosis based on real-time PCR. Twenty-four SBP4 nucleotide sequences obtained matched to B. bovis with a similarity of 97–100%. Of 131 infected animals, 87 (17.2%) were positive for B. bovis while 70 (13.8%) had B. bigemina and 26 (5.1%) were observed to be co-infected with both Babesia species. A total of 61 animals (12.1%) were found to be infected with B. bovis parasites only, while 44 animals (8.7%) had B. bigemina only. Babesia bovis and B. bigemina infections were detected in the three Kwale sub-counties.ConclusionThese findings reveal high prevalence of pathogenic B. bovis in a Kenyan area cutting across a busy transboundary livestock trade route with neighbouring Tanzania. The Babesia multiplex real-time PCR assay used in this study is specific and can detect and differentiate the two Babesia species and should be used for routine B. bovis surveillance to monitor the spread and establishment of the pathogen in other African countries where B. bigemina is endemic. Moreover, these findings highlight the threat of fatal babesiosis caused by B. bovis, whose endemic status is yet to be established.Graphical Abtract

A New Lineage of Perch Rhabdovirus Associated with Mortalities of Farmed Perch.

A perhabdovirus was isolated from a mortality episode affecting a fish farm in 2019 in Western Europe. This virus was produced in cell culture and was readily detected by a species-specific real-time PCR assay. The near-complete sequence of the virus obtained showed some relatedness with viruses of the species Perhabdovirus perca. However, it was distinct enough from these viruses to form a separate genetic lineage. Multiple substitutions along the genome caused non-detection using a range of conventional PCRs previously shown to target four known genogroups of perhabdoviruses. However, various generic PCRs efficiently detected the isolated virus. The origin of this virus remains to be elucidated. It may have been introduced into the farm via wild genitors. This finding provides new evidence of the high genetic diversity of percid perhabdoviruses and the potential of new genotypes to emerge as threats for fish farming. Efforts to improve the existing diagnostic methods and control this large group of viruses are still needed.

Molecular detection and distribution of the genera Amphidoma and Azadinium (Amphidomataceae, Dinophyceae) in the coastal waters of Aotearoa/New Zealand

ABSTRACT Molecular-based approaches for harmful algal bloom species, which can be difficult to identify by light microscopy, are important tools for detecting and predicting the blooms. In this study, 84 samples from Aotearoa/New Zealand coastal waters that contained Azadinium-like cells were analysed using an Amphidomataceae real-time PCR assay. Nineteen samples were positive, and 24 Azadinium-like clonal isolates were established from these samples. Subsequently, only five isolates from one sampling point were positive using the real-time PCR assay. Four of the five isolates were genetically identified as Amphidoma languida and one as Azadinium dalianense. The field samples were analysed retrospectively using the species-specific real-time PCR assays; Am. languida was detected from the temperate areas, while Az. poporum and Az. spinosum were detected at one site. Two representative strains of each species were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), including Am. languida, which is known as toxic in the previous studies, but neither produced the currently monitored azaspiracids (AZAs). These two newly recorded species for New Zealand are important findings. The results highlight the need for more information on the spatio-temporal variations of potentially toxic Amphidomataceae species and the risk of AZAs in New Zealand coastal waters.

Development of eDNA tools for the detection of marine invasive non-native species to support European flat oyster (Ostrea edulis) restoration projects.

The European flat oyster (Ostrea edulis) is an important keystone species in Scottish coastal waters. However due to anthropogenic pressures, significant reductions to oyster beds have been observed across Europe. In Scotland, several projects are currently aiming to restore European flat oyster habitats through the translocation of juvenile oysters from various sources including hatcheries and aquaculture. However, translocation of shellfish is not risk free and can increase the risk of accidental translocation of invasive non-native species (INNS). If INNS become established outside of their native range they can cause irreversible harm to native organisms and habitats. This study aims to develop molecular tools to detect environmental DNA of INNS which can be potentially associated with the translocation of live shellfish stocks. We have developed a species-specific real-time PCR assay for detection of Pacific oyster (Crassostrea gigas) and tested its sensitivity in a large-scale replicated mesocosm based experiment with varying densities of C.gigas. A secondary objective of the experiment was to assess the detection of another invasive species, the carpet sea squirt Didemnum vexillum which was cohabited with C. gigas. We aim to quantify the detection probability of increasing densities of C. gigas from repeat water samples and qPCR replicates. This project also aims to investigate the feasibility of using portable, real-time sequencing technologies such as the Oxford Nanopore MinION to develop robust tools to support native oyster restoration programmes.

Development of a species-specific real-time PCR test for Chlamydia psittaci and its employment in the investigation of zoonotic transmission from racing pigeons in Denmark

Species-specific detection of Chlamydia psittaci is challenging and all published PCR tests have so far shown deficiencies in specificity or sensitivity. The present investigation reports on the development of a species-specific real-time PCR assay for C. psittaci. The test is based on an 84 bp indel in a gene of unknown function that is unique to C. psittaci. The Cps-indel84-PCR assay was validated on a wide range of chlamydial and other bacterial strains as well as on clinical samples from animals and humans in two different diagnostic laboratories in Germany and Denmark. Furthermore, the test was employed for investigating samples from racing pigeon flocks in Denmark. The evaluation showed that the Cps-indel84-PCR assay has excellent test characteristics and is a highly reliable method for identifying C. psittaci in clinical samples both from humans and animals.

Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples.

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

Detection of meat from horse, donkey and their hybrids (mule/hinny) by duplex real-time fluorescent PCR.

Meat adulteration is currently a common practice worldwide. In China, adulteration of donkey meat products with the similar species (horse and mule/hinny) meat and mislabeling are becoming widespread concerns. In this study, a sensitive and species-specific duplex real-time PCR assay based on the simultaneous amplification of fragments of the creatine kinase muscle gene family, was developed and optimized for the identification of horse, donkey and mule /hinny species in raw and heat-processed meat products. Duplex real-time PCR results showed different fluorescence amplification curves for horse and donkey. Both kinds of fluorescence amplification curves appeared simultaneously for mule/hinny. The limit of detection (LOD) of the method was up to 0.01 ng /μL. The method and strategy developed in this study could be applied to detect the presence of adulterants from horse and mule /hinny meat in raw donkey meat and meat products.

The future of biodiversity monitoring and conservation utilizing environmental DNA

The future of biodiversity monitoring and conservation utilizing environmental DNA

Tick-borne rickettsiae in Midwestern region of Republic of Korea

To identify spotted fever group (SFG) rickettsiae among ticks collected by dragging at eight sites in three provinces of the midwestern region of the Republic of Korea (ROK), genus- and species-specific quantitative real-time PCR (qPCR) assays and sequencing were performed. DNA was extracted from a total of 2,312 ticks that were assayed individually (n=140) or in pools (n=444), resulting in a total of 584 individual and pooled tick samples. The 584 tick samples were screened with the genus-specific qPCR assay (Rick17b) and produced 265 (45.38%) positive reactions [individual (n=64) and pooled (n=101) samples]. Of these genus-specific positive samples, 57 (21.51%) were identified as Candidatus Rickettsia longicornii and 48 (18.11%) were identified as R. monacensis by species-specific qPCR assays. Subsequently, nested PCR (nPCR) was performed with 120 samples, which tested positive samples for genus-specific, but not species-specific, qPCR assays. The sequences of ompA and ompB genes showed how many close relatedness to Ca. R. longicornii and Ca. R. jingxinensis isolate Xian Hl-79, uncultured Rickettsia sp. Y27-1, Ca. R. tasmanensis strain T152, R. endosymbiont of H. longicornis tick 47, and R. koreansis strain CNH17-7. In conclusion, we successfully detected specific rickettsial agents using qPCR and a sequence-based analysis approach that demonstrated the prevalence of various tick-borne Rickettsia spp. in midwestern ROK.

Spotted Fever Group Rickettsia Infection of Cats and Cat Fleas in Northeast Thailand.

Rickettsia species cause rickettsioses, which are zoonotic diseases found worldwide, and are transmitted by arthropods such as lice, fleas, ticks, and mites. In Thailand, flea infestations are common among cats and dogs. This study aimed at determining the exposure to spotted fever group rickettsiae (SFGR) of cats in surrounding areas of Rajabhat Maha Sarakham University, Muang district, Maha Sarakham province and rickettsial infection among cat fleas, Ctenocephalides felis, collected from dogs of the surrounding area of Waeng Noi district, Khon Kaen province. Forty-two cat sera were assessed for IgG antibody titers against SFGR by a group-specific enzyme-linked immunosorbent assay. The prevalence of seroreactive cats was 4.76% (2/42). DNA preparations from 23 individual cat fleas from three dogs were assessed by Rickettsia genus-specific, group-specific, and species-specific quantitative real-time PCR (qPCR) assays. Positive results were confirmed by ompB gene fragment sequencing. Twenty-one of 23 cat fleas were positive for Rickettsia asembonensis, and the other two DNA preparations were negative for rickettsial DNA. This study's finding indicates that companion cats and dogs in Northeast Thailand are exposed to SFGR and that exposure may be due to infection with R. asembonensis, an organism known to infect humans, monkeys, and dogs. Clinicians for humans and animals in Northeast Thailand should be aware of rickettsial infections among their patients.

A diagnostic real-time PCR assay for the rapid identification of the tomato-potato psyllid, Bactericera cockerelli (Šulc, 1909) and development of a psyllid barcoding database.

The accurate and rapid identification of insect pests is an important step in the prevention and control of outbreaks in areas that are otherwise pest free. The potato-tomato psyllid Bactericera cockerelli (Šulc, 1909) is the main vector of 'Candidatus Liberibacter solanacearum' on potato and tomato crops in North America and New Zealand; and is considered a threat for introduction in Europe and other pest-free regions. This study describes the design and validation of the first species-specific TaqMan probe-based real-time PCR assay, targeting the ITS2 gene region of B. cockerelli. The assay detected B. cockerelli genomic DNA from adults, immatures, and eggs, with 100% accuracy. This assay also detected DNA from cloned plasmids containing the ITS2 region of B. cockerelli with 100% accuracy. The assay showed 0% false positives when tested on genomic and cloned DNA from 73 other psyllid species collected from across Europe, New Zealand, Mexico and the USA. This included 8 other species in the Bactericera genus and the main vectors of 'Candidatus Liberibacter solanacearum' worldwide. The limit of detection for this assay at optimum conditions was 0.000001ng DNA (~200 copies) of ITS2 DNA which equates to around a 1:10000 dilution of DNA from one single adult specimen. This assay is the first real-time PCR based method for accurate, robust, sensitive and specific identification of B. cockerelli from all life stages. It can be used as a surveillance and monitoring tool to further study this important crop pest and to aid the prevention of outbreaks, or to prevent their spread after establishment in new areas.

Molecular surveillance of anti-malarial drug resistance in Democratic Republic of Congo: high variability of chloroquinoresistance and lack of amodiaquinoresistance

BackgroundThe loss of chloroquine (CQ) effectiveness has led to its withdrawal from national policies as a first-line treatment for uncomplicated malaria in several endemic countries, such as the Democratic Republic of Congo (DRC). The K76T mutation on the pfcrt gene has been identified as a marker of CQ resistance and the SVMNT haplotype in codons 72–76 on the same gene has been associated with resistance to amodiaquine (AQ). In the DRC, the prevalence of K76T has decreased from 100% in 2000 to 63.9% in 2014. The purpose of this study was to determine the prevalence of K76T mutations in circulating strains of Plasmodium falciparum, 16 years after CQ withdrawal in the DRC and to investigate the presence of the SVMNT haplotype.MethodsIn 2017, ten geographical sites across the DRC were selected. Dried blood samples were collected from patients attending health centres. Malaria was first detected by a rapid diagnostic test (RDT) available on site (SD Bioline Malaria Ag Pf or CareStart Malaria Pf) or thick blood smear and then confirmed by a P. falciparum species-specific real-time PCR assay. A pfcrt gene segment containing a fragment that encodes amino acids at positions 72–76 was amplified by conventional PCR before sequencing.ResultsA total of 1070 patients were enrolled. Of the 806 PCR-confirmed P. falciparum positive samples, 764 were successfully sequenced. The K76T mutation was detected in 218 samples (28.5%; 95% CI 25.4%–31.9%), mainly (96%) with the CVIET haplotype. Prevalence of CQ resistance marker was unequally distributed across the country, ranging from 1.5% in Fungurume to 89.5% in Katana. The SVMNT haplotype, related to AQ resistance, was not detected.ConclusionOverall, the frequency of the P. falciparum CQ resistance marker has decreased significantly and no resistance marker to AQ was detected in the DRC in 2017. However, the between regions variability of CQ resistance remains high in the country. Further studies are needed for continuous monitoring of the CQ resistance level for its prospective re-use in malaria management. The absence of the AQ resistance marker is in line with the use of this drug in the current DRC malaria treatment policy.