A multiplex PCR for the identification of Echinococcus multilocularis, E. granulosus sensu stricto and E. canadensis that infect human

Abstract

Echinococcus granulosus sensu stricto (s.s.), Echinococcus multilocularis and Echinococcus canadensis are the common causes of human echinococcosis in China. An accurate species identification tool for human echinococcosis is needed as the treatments and prognosis are different among species. The present work demonstrates a method for the simultaneous detection of these three Echinococcus species based on multiplex polymerase chain reaction (mPCR). Specific primers of this mPCR were designed based on the mitochondrial genes and determined by extensive tests. The method can successfully detect either separated or mixed target species, and generate expected amplicons of distinct size for each species. Sensitivity of the method was tested by serially diluted DNA, showing a detection threshold as less as 0.32 pg for both E. granulosus s.s. and E. canadensis, and 1.6 pg for E. multilocularis. Specificity assessed against 18 other parasites was found to be 100% except weakly cross-react with E. shiquicus. The assay was additionally applied to 69 echinococcosis patients and 38 healthy persons, confirming the high reliability of the method. Thus, the mPCR described here has high application potential for clinical identification purposes, and can further provide a useful tool for evaluation of serology and imaging method.

Plasma Circulating Cell-Free DNA Facilitated the Detection of an Alveolar Echinococcosis Patient Initially Misdiagnosed as Cystic Echinococcosis: A Case Report

“…Though some Echinococcus cfDNA reads could not be grouped into specific species (4/49 in our case), the majority of the Echinococcus cfDNA reads could be classified into the correct species, and thus could be used to facilitate the diagnosis. The advantage of using plasma cfDNA to facilitate the diagnosis is that the result was molecular-based and more objective and could be obtained before the surgery, thus different from a lot of the other PCR-based molecular methods using tissue samples which could only be obtained after the surgery [ 15 , 19 , 20 ].…”

“…To facilitate the accurate diagnosis of human echinococcosis, different methods were developed. For example, Oberli, Chen, and Shang’s respective research teams developed multiplex polymerase chain reaction (PCR)-based methods that could differentiate between AE and CE using biopsy materials or tissue/lesion samples [ 15 , 19 , 20 ]. As not all echinococcosis patients would undergo surgery, tissue/lesion/biopsy materials are not always available.…”

Advances in the study of molecular identification technology of Echinococcus species

“…Multiplex PCR is widely used in the differential detection of Echinococcus. It can simultaneously detect multiple species of Echinococcus in human or various animals infected samples, which makes this method fast, efficient and economical (Chen et al, 2019;Shang et al, 2019). Multiplex PCR allows species identification from cysts or stool specimens.…”

“…When it comes to the species identification, simultaneous detection of E. granulosus, E. multilocularis, and E. Canadensis (E. granulosus subtype) based on multiplex PCR, reporting that the detection threshold for E. granulosus and Echinococcus canadensis was 0.32 pg and 1.6 pg for E. multilocularis. In addition to the weak cross reaction with E. shiquicus, the specificity for the other 18 parasites was 100% (Chen et al, 2019). In the genotyping research, multiplex PCR was used to identify E. granulosus species except G1 and G3 genotypes, which has been developed and verified in the European Union Parasite Reference Laboratory (EURLP) (Santolamazza et al, 2020).…”

Study on the mechanism of miRNAs on liver injury in the condition of Protoscocephalus alveolarus transhepatic portal vein infection

“…The most common site of disease is mainly in the liver, and a few can also be found in the lungs and other organs 5,6 . The prevalence and incidence of CE is higher than AE 7,8 . But the damage of CE to the body is significantly higher than that of AE.…”