Rapid Detection Of EHP Using Taqman Probe QPCR And Its Growth Correlation With Shrimp Samples

In recent years, the phenomenon of slow growing in prawn has been reported more and more in shrimp farms in Southeast Asian. The prawn cultured in these farms were seriously infected with one microsporidian, namely Enterocytozoon hepatopenaei(EHP), which was firstly found in the slow growing Penaeus monodon cultured in Thailand. EHP, an intracellular parasite, belongs to the family Nosematidae, the genus Enterocytozoon and duplicates in the cytoplasm of the epithelial cells of hepatopancreas in the host. No obvious clinical symptoms have been observed in EHP infection and the diagnosis of this phenomen is mainly through histological observation, PCR assay, digoxigenin labeled probe in situ hybridization assay, and LAMP detection assay, however, these detection assays could only qualitative describe that pathogenic microorganism without carrying out quantitative research.SYBR Green I qPCR assay was established in this laboratory to detect EHP in earlier stage. This method has relative higher detection sensitivity and realizes the quantitative detection of samples, as well as provides technical means for the detection, prevention and control of EHP. Among the quantitative detection methods, specific probe is used in the Taqman probe assay which can detect the target sequence with very strong specificity. Therefore, the Taqman probe assay could effectively prevent the influence caused by the nonspecific products in PCR reaction and further improve the accuracy of quantitative assay, and has been extensively applied in the detection of aquatic animal pathogens. In the OIE aquatic animal diagnosis standard of shrimp pathogen, the Taqman probe assay often becomes the standard methods for quantitative detection.Taqman probe qPCR assay for EHP was established in this study. It showed a good linearity in detecting standards of EHP SSU rDNA from 6.1×101 to 6.1×108 copies/ μL using the established method, and good intra-assay and inter-assay repeatability were observed within the linearity range. From the perspective of detecting farm samples, comparing with the nested PCR(Amornrat et al., 2013) and SYBR Green I qPCR-EHP, the sensitivity of the assay established in this study was higher than nested PCR and SYBR Green I qPCR-EHP by 7.6±4.2 times and 1.3±0.2 times. Therefore in detecting the farm samples, the positive detection rate of Taqman qPCR-EHP(93.5%) was obviously higher than that of nested PCR(77.4%), and was a little higher than that of SYBR Green I qPCR-EHP(88.7%); comparing with Taqman qPCR-EHP, the diagnostic specificity of nested-PCR and SYBR Green I q PCR –EHP was 100% and the diagnostic sensitivity were 82.8% and 96.6%, respectively, indicating Taqman qPCR-EHP had a higher diagnostic sensitivity. The correlation of the quantitative results of Taqman qPCR-EHP and SYBR Green I qPCR-EHP was high and the value comparability was good. Although SYBR Green I flurochrome method is applied to the quantitative analysis of DNA, the lack of specific fluorescent probe could cause the interference of primer dimer and non-specific amplification products, thereby affect the accuracy of results even false positive. That method has rarely been used as a standard method for quantitative detecting pathogens in OIE aquatic animal diagnostic standards. Therefore, SYBR Green I qPCR assay was established in this laboratory to detect EHP in earlier stage, however, Taqman qPCR assay was established on that basis in this study. Through the comparison, method established in this study had a higher sensitivity and specificity than SYBR Green I and nested PCR, which could guarantee the sensitivity and accuracy of quantitative determination of EHP.Body length and weight of the four batches of L. vannamei collected from Tianjin and Zhejiang were measured individually in this study, and BMI was calculated. Total DNA sample of each shrimp hepatopancreas was extracted and Taqman qPCR-EHP was used to detect EHP copies in hepatopancreas DNA samples. The result showed that EHP copies had a negative correlation with BMI in the samples from Tianjin, namely EHP had an effect on the growth status of L. vannamei, which was consistent with the conclusion we drawn from the three batches of samples collected from Danzhou in Hainan, Jimo in Shandong and Ganyu in Jiangsu by SYBR Green I qPCR(Liu et al., 2016). And it was also proved directly by Taqman qPCR-EHP assay that EHP copies appeared obvious decrease in the two batches of samples collected before and after some kind of treatment on EHP, the logarithmic mean value of EHP copies after the treatment fell to(48.4±39.4) % of the value before the treatment, indicating that qPCR method established in this study could be used in the evaluation of EHP treatment effect. However, it should be worth noting that the correlation of EHP copies of the shrimp collected in the cultured pond and the BMI was not always negative. In the other two batches of samples collected from Tianjin and Zhejiang, EHP copies had no obvious correlation with BMI, and it also had no obvious correlation with the body length and weight, but these individuals of L. vannamei with slow growth were all EHP positive. Low correlation was also observed in several samples with long body length detected by SYBR Green I q PCR in our previous studies, it was indicated that EHP infection could affect the growth of L. vannamei while the change in EHP copies in hepatopancreas might not always have a correlation with the body length. It was speculated that there were some differences in the time of EHP infection in different individuals of L. vannamei, and unknown mechanisms might exist in the stages of EHP infection process affecting the growth of L. vannamei which need to be further studied in the future.Microsporidia Enterocytozoon hepatopenaei(EHP) was detected with Taqman probe based real time PCR for 442 shrimp from 5 Litopenaeus vannamei populations in farms in Tianjin, Zhejiang, and Shandong. And the biological length and body weight of each shrimp were measured individually. The Rohrer’s ponderal index(Pi, W/L3) used in medical science was introduced to establish the fuction of body weight(W) and biological length(L) of shrimp. The results showed that the Pi of 4 EHP positive populations with an average biological length at 5.37±1.19 cm was counted as(5.19±0.26)×10-3 g/cm3, while that of the EHP negative population with an average biological length was(7.96±0.51)×10-3 g/cm3. After adjusting of the difference of Pi caused by biological length according to the function Pi=a L(b-3), the Pi of EHP positive populations was(70.5±8.7)% of that of the EHP negative populations at the same biological length. It indicated that the average body weight of shrimp in an EHP positive population may less by about 30% than that of shrimp with same size in biological length in an EHP negative population. The coefficient of variation(C.V.) of biological length and body weight in EHP positive populations were 2.39±0.93 times and 2.05±0.86 times of those in the EHP population, respectively, which shows significant size variation in EHP positive populations. The deviation ratio of actual body weight to body weight estimated on biological length in EHP positive populations was 2.34—3.45 times of that in the EHP negative population, which shows the body weight of the shrimp with same size in EHP positive population had greater unexpected fluctuation.