A Field-Deployable Insulated Isothermal PCR (iiPCR) for the Global Surveillance of Toxoplasma gondii Infection in Cetaceans

Hsieh, Meng-Jung; Yang, Wei-Cheng

doi:10.3390/ani12040506

Cited by 7 publications

(3 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cats are not only the definitive host of T. gondii but also excrete the environmentally resistant oocysts in their feces ( 10 ). Millions of T. gondii oocysts can be shed from felids, leading to infection of other susceptible hosts ( 11 ). Meta-analyses over the past 50 years showed that the seroprevalence of T. gondii in domestic and wild felids was around 35% and 59%, respectively ( 12 ).…”

Section: Introductionmentioning

confidence: 99%

RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect Toxoplasma gondii in stray dogs and cats in Zhejiang province, China

Sun,

Fan,

Chu

et al. 2024

Microbiol Spectr

View full text Add to dashboard Cite

Toxoplasma gondii , which causes toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. T. gondii causes economic losses to livestock production and represents a potential risk to public health. Dogs and cats are common hosts in the epidemiology of toxoplasmosis. The current molecular diagnostic tools for T. gondii infection require high technical skills, a laboratory environment, and complex instruments. Herein, we developed a recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) assay to detect T. gondii . The lowest limit of detection of the assay was 31 copies/μL for the T. gondii B1 gene. In addition, we established a visual RPA-CRISPR/Cas12a lateral flow band assay (RPA-CRISPR/Cas12a-LFA) combined with a digital visualization instrument, which minimized the problem of false-negative results for weakly positive samples and avoided misinterpretation of the results by the naked eye, making the LFA assay results more accurate. The assay established in this study could identify T. gondii within 55 min with high accuracy and sensitivity, without cross-reaction with other tested parasites. The developed assay was validated by establishing a mouse model of toxoplasmosis. Finally, the developed assay was used to investigate the prevalence of T. gondii in stray cats and dogs in Zhejiang province, Eastern China. The positive rates of T. gondii infection in stray cats and dogs were 8.0% and 4.0%, respectively. In conclusion, the RPA-CRISPR/Cas12a-LFA is rapid, sensitive, and accurate for the early diagnosis of T. gondii , showing promise for on-site surveillance. IMPORTANCE Toxoplasma gondii is a virulent pathogen that puts millions of infected people at risk of chronic disease reactivation. Hosts of T. gondii are distributed worldwide, and cats and dogs are common hosts of T. gondii . Therefore, rapid diagnosis of early T. gondii infection and investigation of its prevalence in stray dogs and cats are essential. Here, we established a visual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a-assay combined with a lateral flow band assay and a digital visualization instrument. Detailed analyses found that the assay could be used for the early diagnosis of T. gondii without false-negative results. Moreover, we detected the prevalence of T. gondii in stray cats and dogs in Zhejiang province, China. Our developed assay provides technical support for the early diagnosis of T. gondii and could be applied in prevalence surveys of T. gondii in stray dogs and cats.

show abstract

Section: Introductionmentioning

confidence: 99%

RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect Toxoplasma gondii in stray dogs and cats in Zhejiang province, China

Sun,

Fan,

Chu

et al. 2024

Microbiol Spectr

View full text Add to dashboard Cite

show abstract

“…This technology duplicates the process of in vitro DNA synthesis through enzymatic reaction, may amplify the target gene fragment exponentially, and has the benefits of high sensitivity, good specificity, cheap cost, and fast speed, making it extensively employed in pathogen detection. In less than 40 years since 1985, advanced methods such as nested PCR [ 11 ], multiplex PCR [ 12 ], real-time PCR (qPCR) [ 13 ], droplet digital PCR (ddPCR) [ 14 ], nanoPCR [ 15 ], and insulated isothermal PCR (iiPCR) [ 16 ] have been invented for clinical testing based on following the principles of the technique.…”

Section: Introductionmentioning

confidence: 99%

Development and Application of nanoPCR Method for Detection of Feline Panleukopenia Virus

Xue

Yang

Gao

et al. 2023

Veterinary Sciences

View full text Add to dashboard Cite

Feline panleukopenia (FP) is a severe viral illness caused by the feline panleukopenia virus (FPV), putting sectors like companion cat breeding and endangered feline conservation at risk. The virus has a high morbidity and fatality rate and is found all over the world. We created a novel FPV assay using nanoPCR technology and assessed the method’s specificity and sensitivity. The approach amplified a 345 bp nucleic acid fragment with a minimum detection limit of 7.97 × 102 copies/μL, which is about 100 times greater than traditional PCR. We collected anal swabs from 83 cats suspected of FPV infection for practical application, and the FPV-positive rate determined by the nanoPCR approach was 77.1%. In conclusion, the approach is more sensitive than conventional PCR and more convenient and cost-effective than qPCR methodology and may be utilized for the clinical detection of FPV.

show abstract

“…Few cases of protozoan Toxoplasma gondii infection in cetaceans have been documented in several nations in North America, Western Europe, and Oceania. However, there remains a lack of information regarding this infection in certain regions, including Southeastern Asia, South Asia, South America, and Africa (6). However, a study indicated that there is a plausible chance of toxoplasmosis in cetacean populations in the Philippines by detecting 71% of the sampled cetaceans that were infected with T. gondii (7).…”

Section: Introductionmentioning

confidence: 99%

First report of Strongylidae nematode from pilot whale (Globicephala macrorhynchus) by molecular analysis reveals the cosmopolitan distribution of the taxon

Junsiri,

Islam,

Thiptara

et al. 2023

Front. Vet. Sci.

View full text Add to dashboard Cite

This study investigates the identification, genetic composition, and placement in the evolutionary tree of a particular nematode species found in a short-finned pilot whale in the Gulf of Thailand. To accomplish this, we utilized various methods, including microscopic observations, molecular techniques, and comparative analyses to better understand the characteristics of this parasite. Initially, we concentrated on studying the 18s rDNA sequence through nested PCR, resulting in a 774-bp product. After conducting a BLASTn analysis, we discovered that there were only a few sequences in the GeneBank that shared similarities with our nematode, particularly with Cyathostomum catinatum, although the percent identity was relatively low. To confirm the uniqueness of our sequence, we constructed a phylogenetic tree that demonstrated a distinct branch for our nematode, suggesting significant genetic differentiation from C. catinatum. Additionally, we sequenced a 399-bp section of the ITS2 gene using PCR, and the resulting data showed a close association with the Strongylidae family, specifically with Cylicocyclus insigne. This was further confirmed by BLASTn and CD-HIT-est results, which indicated a 99 and ~94% sequence homology with C. insigne, respectively. The ITS2 phylogenetic tree also supported the position of our isolated sequence within the Strongylidae family, clustering closely with C.insigne. Our findings shed light on the genetic connections, taxonomy, and evolutionary trends within the Strongylidae family, with a particular focus on the widespread nature of the Cylicocyclus genus. This study emphasizes the importance of utilizing molecular techniques and interdisciplinary approaches to gain insight into nematode diversity, evolution, and ecological dynamics in marine environments.

show abstract

A Field-Deployable Insulated Isothermal PCR (iiPCR) for the Global Surveillance of Toxoplasma gondii Infection in Cetaceans

Cited by 7 publications

References 58 publications

RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect Toxoplasma gondii in stray dogs and cats in Zhejiang province, China

RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect Toxoplasma gondii in stray dogs and cats in Zhejiang province, China

Development and Application of nanoPCR Method for Detection of Feline Panleukopenia Virus

First report of Strongylidae nematode from pilot whale (Globicephala macrorhynchus) by molecular analysis reveals the cosmopolitan distribution of the taxon

Contact Info

Product

Resources

About