| Background: with the development of globalization,pathogens spread and spread all over the world,resulting in a large number of mixed infections of various pathogens in animals,which has a great impact on the development of agricultural production.With the gradual complication of mixed infection,sick animals may be infected with a variety of pathogens with similar symptoms at the same time.The existing conventional detection met hods are timeconsuming and labor-consuming.As the detection met hod with the largest flux among the existing conventional detection met hods,multiplex PCR detection technology can detect up to four pathogens at the same time,which is difficult to cover all possible pathogenic pathogens.Therefore,it is particularly important to develop a high-throughput nucleic acid detection technology.Objective: the purpose of this study is to develop a high-throughput nucleic acid detection technology based on probe fusion curve by using the high specificity of molecular beacon and the met hod of introducing label on amplification products by bridge amplification.Met hods: a group of tags and molecular beacons with TM value difference above 5℃were designed as tags and molecular beacons for single channel multiple detection.According to the nucleic acid sequence of each pathogen published by NCBI nucleoside,each pathogen specific primer is designed.By adding a base sequence in front of the 5 ’end of the upstream primer,the tag complementary sequence is introduced into the amplified nucleic acid sequence,and the tag homologous molecular beacon is used as a probe to detect the tag complementary sequence.According to the TM value corresponding to the melting curve peak in the results of melting curve analysis,the type of pathogen in the sample is determined.Using the high specificity of the improved molecular beacon,based on the designed label and molecular beacon,new labels and molecular beacons are obtained through the exchange of single or multiple bases of A / T and C / G,which can be used as labels and molecular beacons of other channels.There was no significant change in the molecular beacon TM value after base replacement.In this study,TGEV,PEDV,CSFV,ASFV,general FMDV,porcine RVs,highly pathogenic PRRSV and PCV2 were taken as examples to verify the method.Results:(1)single channel multiple detection was realized based on the difference of molecular beacon fusion curve: the purpose of multiple detection in fam channel was realized by setting label sequences with different TM values.(2)Two dimensional PCR detection technology based on molecular beacon fusion curve:in this study,a tag and probe sequence library for two-color fluorescence channels was established,and eight targets such as porcine RVs,highly pathogenic PRRSV and PCV2 were successfully detected by two-dimensional PCR.(3)Application and evaluation of two-dimensional PCR detection technology of important animal pathogens: the sensitivity test of fluorescence quantitative PCR with the same probe met hod as the specific primer showed that there was no significant difference between the two met hods.the consistency analysis of clinical samples shows that the kappa coefficient of the two met hods is 0.98,which is greater than 0.75,so the two met hods are highly consistent.Conclusion: the two-dimensional PCR the technology of molecular beacon combined with label has good consistency with probe fluorescence quantitative PCR.It is suitable for the detection of high-throughput and multi-target,the detection of mixed infection of multiple pathogens,the typing of pathogenic microorganisms and the identification of multiple drugresistant bacteria. |
