Use Of A Universal Hydroxymethylbilane Synthase(HMBS)Based SYBR Green-qPCR As An Endogenous Internal Control For Brids DNAs And Its Application

Since 1985,polymerase chain reaction(PCR)technology has been widely used in medicine and Molecular biology due to its high sensitivity,specificity and fast detection.The accuracy of the PCR results largely depends on the quality of the sample nucleic acid to be examined.However,the samples used for PCR detection were affected by human and objective factors in the process of collection,transportation,storage and so on,therefore,it is very important toestablish the nucleic acid quality control system.Currently,the quality control system used for molecular detection of birds PCR has only exogenous control system,which has great defects than endogenous internal control(EIC)system.In this study,an EIC system was established to detect nucleic acids of all bird's species.This system serves as quality monitoring in the whole process from samples collection to PCR detection of bird's nucleic acids.1.Establishment of an endogenous internal control system for birds DNA based on HMBS SYBR Green-qPCR.In the first study,SYBR Green quantitative PCR system for the hydroxymethylbilane synthase(HMBS)gene of birds was designed,the birds HMBS gene is an internal reference gene of the tissue or organ of birds.The endogenous internal control system established in this experiment could detect the nucleic acid of 8 kinds of birds'(chicken,duck,goose,pigeon,wild goose,swan,crane,quail)throat swab and 11 kinds of tissues or organs of chickens or seven local breeds of chicken(black-shelled layer chickens,white strips,reed chickens,rugao yellow chickens,xianju chickens,beard chickens,bluegill chickens,white-eared chickens,gaoju chickens),but not amplification nucleic acid from mammals,parasites,arthropods,and bacteria.The bird's endogenous internal control system has high sensitivity(a single copy/reaction)and specificity(only amplifies the nucleic acids of birds).2.Application of an endogenous internal control system of birds DNA based on HMBS SYBR Green-qPCR.According to the same sampling method,sample preservation solution and transportation method,different samplers collected 415 samples of poultry throat samples from the live poultrymarkets in five provinces(Jiangsu,Yunnan,Shandong,Fujian,and Hubei)and the Tibet Autonomous Region,the samples were detected for PCR detection of two pathogens(avian influenza virus,chlamydia),colistin resistance gene mcr-1.In the meantime,the bird's endogenous internal control system established in this study was used to evaluate the quality of collected samples.The positive rates of the two pathogens and the colistin resistance gene mcr-1 in six regions were significantly different.The highest positive rate of AIV was 31.4% in Yunnan province,followed by 26.1% in Jiangsu,14.7% in Fujian province,the positive rate of AIV in Shandong,Hubei and Tibet were 0%.The highest positive rate of Chlamydia was 67.1% in Yunnan Province,followed by 50% in Shandong Province,29.4% in Fujian Province,10% in Jiangsu Province,1.6% in Hubei Province,and 0% in Tibet.The monitoring results of HMBS SYBR Green-qPCR method showed that the proportion of positive HMBS gene in the three provinces of Shandong,Jiangsu and Yunnan were 100/100(100%),the average copy number of HMBS gene were 10 5.15±0 97[SD]/swab,10 4.59±0.35 [SD]/swab and 10 4 57±0.49 [SD]/swab,However,the HMBS genes were not detected in 2,15 and 11 samples from Hubei,Fujian and Tibet respectively,the average HMBS gene copy number were 10 3 59±0 39[SD]/swab,10 3.84±0.69[SD]/swaband 103.01±1.11[SD]/swab.the internal control system established in this study can accurately monitor the quality of nucleic acids in clinical samples of birds,filter out failed samples to significantly reduced the false negative rate of PCR detection.3.Validation of an endogenous internal control system for birds DNA based on HMBS SYBR Green-qPCR.To prove that the system can be applied to accurately monitor nucleic acids quality of samples,in this study,20 SPF chickens were infected with avian influenza virus,the throat swabs were collected using three sampling methods(Hard,Middle,Gentle),and were stored in three protective solutions(DNA/RNA nucleic acid stabilizer,1×PBS,and four antibiotics mixture).in addition,those samples were placed at room temperature(0 days,3 days and 9 days),all samples were subsequently tested for HMBS SYBR Green-qPCR and based on 23 S rRNA qPCR for avian influenza virus.The test results showed that:l)samples hard-collection were all positive for HMBS gene detection,and copy number of HMBS gene of 20 samples were higher than the lowest detection limit of pathogens,while the positive rate of H9N2 AIV detected was90%.2)The positive rates of HMBS gene of samples Middle-collection and samples Gentle-collection were 50% and 20% respectively.while the positive rates of H9N2 AIV were50% and 0% respectively.The samples with HMBS gene copies lower than 10 3.70±0.4[SD]/swab did not detected H9N2 AIV.The DNA/RNA nucleic acid stabilizer was the best in three sample protection solutions,and there was no effect on the positive rate of H9N2 AIV when the samples were preserved up to 9 days at room temperature.The positive rate of pathogens dropped significantly for two other protection solutions stored at room temperature for 3 days or more.This study verified that the sampling method,sample protection solution and room temperature storage time have a great influence on sample nucleic acid levels and quality,thus affecting the positive detection rate of pathogens.In conclusion,HMBS SYBR Green-qPCR endogenous internal control system for bird's DNA established in this study is capable of monitoring the quality of clinical sample nucleic acids with great sensitivity and specificity.It is the monitoring and verification of the entire process of collection,transportation,preservation,nucleic acid extraction and PCR detection of bird samples.This approach can monitor the quality of the bird's clinical samples in real time and provide a quality control system for molecular detetection of avian infectious agents and other antimicrobial resistance genes.