The Exploration Of Random Cloning Technology For Suspicious Pathogen Nucleic Acid In Suffering Macrobrachium Rosenbergii Larvae

Macrobrachium rosenbergii, belongs to the family Palaemonidae，Macrobrachium,also known as the genus of white leg shrimp, Malaysia prawn shrimp, Wan prawn,originating in the India Pacific region, living in various types of freshwater or brackishwaters, introduced to our country from Japan in1976, is one of three prawn breed witha highest quantity in the present world and a basically breed variety of freshwatercrustacean in China. But in recent years, with the continuous expansion ofMacrobrachium rosenbergii culture area, ceaselessly intensive rising degree, virusdisease is also frequent, bringing great threat for Macrobrachium rosenbergii culture. Sofar, the reported Macrobrachium rosenbergii virus has four types: WSSV,Macrobrachium rosenbergii Nodavirus(MrNV)causing Macrobrachium rosenbergiiwhitish muscle diseases (whitish muscle disease, WTD)and XSV(extra small virus-likeparticles)may serving as a MrNV satellite virus or helper virus tiny virus, as well as arelatively rare parvo-like virus infecting epithelial cells of Macrobrachium rosenbergiiliver pancreas (parvo-like virus).In March2011, we collected a number of diseased larvae samples in a giantfreshwater prawn culturing plant from Zhejiang. By homogenate,0.45micronmembrane filtration, centrifugation, DNase I and RNase A processing, extraction ofsuspicious viral nucleic acid, we use multiple random PCR and sequence independentsingle primer amplification(SISPA) for DNA and RNA random cloning and sequencing,analysis sequencing results by BLASTn, specific detection for suspicious sequence.Through multiple random PCR technology on the suspicious virus DNA analysis,we obtain22sequencing results, using the NCBI BLASTn in the NR database forcomparison, and14of these clones are vectors,1clone result is meaningless and2clones are bacteriral sequences,3clones are Macrobrachium rosenbergii genomesequences,2of them are suspicious low homology sequence, the minimum sequenceis45bp, the maximum is1135bp, the average length is873bp.2suspicious viralsequences are597bp and1053bp respectively.Through the sequence of independent single primer amplification (SISPA)technology on the suspicious virus RNA analysis, we received a total of48suspectedinserted DNA clonal sequence origininal RNA extract, use NCBI BLASTN function inthe NR library for comparison,7of these clones are vector,17clones results arenonsense,10clones are bacteria.5clones are Macrobrachium rosenbergii genome sequences or mitochondrial gene sequences,2clones are suspicious viral sequences,7clones are low homology sequences. The length of shortest sequence is71bp, thelongest length is1167bp, the average length is389bp. In the two section and sequencingof virus that is part of homologous with Passiflora chlorotic virus inserted clonesequences, a long551bp cloned fragment and (Passiflora chlorosis virus, PCV)sequences have certain homology, a long441bp cloned fragment and rotavirus(Rotavirus, RV) sequences certain homology. According to the two inserted fragmentsequences, we designed primers for sample specific identification of these clonedfragment, and it showed that the two sequences are derived from the collection ofMacrobrachium larve samples.In this paper, the use of multiple random PCR asequence-independent singleprimer amplification for analysis results of Macrobrachium rosenbergi samples showthat the two kinds of random clone technology can be applied to suspicious virusidentification and suspicious pathogen identification, epidemic monitoring, and they canplay important role in the early prevention of diseases on aquatic animal breeding.