Preliminary application of recombinase -aided amplification in detection of Clonorchis sinensis metacercariae in freshwater fish.
10.16250/j.32.1374.2023020
- Author:
J CHEN
1
;
Z WANG
1
;
W HUANG
1
;
J WANG
1
;
L CHEN
1
;
Y SUN
1
;
L ZHAO
2
;
Y ZHAO
3
;
Y QIAN
4
;
J DUAN
5
;
Q ZHANG
6
Author Information
1. Taizhou Municipal Center for Disease Control and Prevention, Taizhou, Jiangsu 225300, China.
2. Taixing Center for Disease Control and Prevention, Taizhou City, Jiangsu Province, China.
3. Hailing District Center for Disease Control and Prevention, Taizhou City, Jiangsu Province, China.
4. Jiangyan District Center for Disease Control and Prevention, Taizhou City, Jiangsu Province, China.
5. Xinghua Center for Disease Control and Prevention, Taizhou City, Jiangsu Province, China.
6. National Health Commission Key Laboratory of Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Jiangsu Institute of Parasitic Diseases, Wuxi, Jiangsu 214064, China.
- Publication Type:Journal Article
- Keywords:
Detection efficiency;
Freshwater fish;
Metacercaria;
Recombinase-aided amplification
- MeSH:
Animals;
Clonorchis sinensis/genetics*;
Metacercariae/genetics*;
Recombinases;
Fresh Water;
Fishes;
DNA;
Fish Diseases/diagnosis*
- From:
Chinese Journal of Schistosomiasis Control
2023;35(5):458-463
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To evaluate the performance of recombinase-aided amplification (RAA) assay in detection of Clonorchis sinensis metacercariae in freshwater fish samples, so as to provide insights into standardization and field application of this assay.
METHODS:Wild freshwater fish samples were collected in the rivers of administrative villages where C. sinensis-infected residents lived in Jiangyan District, Xinghua County and Taixing County of Taizhou City, Jiangsu Province from June to September 2022. Genomic DNA was extracted from six freshwater fish specimens (5 g each) containing 0, 1, 2, 4, 8 and 16 C. sinensis metacercariae for fluorescent RAA assay, and the diagnostic sensitivity was evaluated. Fluorescent RAA assay was performed with genomic DNA from C. sinensis, Metorchis orientalis, Haplorchis pumilio and Centrocestus formosanus metacercariae as templates to evaluate its cross-reactions. In addition, the detection of fluorescent RAA assay and direct compression method for C. sinensis metacercariae was compared in field-collected freshwater fish samples.
RESULTS:Positive amplification was found in fresh-water fish specimens containing different numbers of C. sinensis metacercariae, and fluorescent RAA assay was effective to detect one C. sinensis metacercaria in 5 g freshwater fish specimens within 20 min. Fluorescent RAA assay tested negative for DNA from M. orientalis, H. pumilio and C. formosanus metacercariae. Fluorescent RAA assay and direct compression method showed 5.36% (93/1 735) and 2.88% (50/1 735) detection rates for C. sinensis metacercariae in 1 735 field-collected freshwater fish samples, with a statistically significant difference seen (χ2 = 478.150, P < 0.001). There was a significant difference in the detection of C. sinensis metacercariae in different species of freshwater fish by both the direct compression method (χ2 = 11.20, P < 0.05) and fluorescent RAA assay (χ2 = 20.26, P < 0.001), and the detection of C. sinensis metacercariae was higher in Pseudorasbora parva than in other fish species by both the direct compression method and fluorescent RAA assay (both P values < 0.05).
CONCLUSIONS:Fluorescent RAA assay has a high sensitivity for detection of C. sinensis metacercariae in freshwater fish samples, and has no cross-reactions with M. orientalis, H. pumilio or C. formosanus metacercariae. Fluorescent RAA assay shows a higher accuracy for detection of C. sinensis infections in field-collected freshwater fish than the direct compression method.
