Study On The Association Between Bovine Leukemia Virus And Human Breast Cancer

Bovine leukemia virus(BLV)infection is reported worldwide,including in China.As reported,BLV infections may even induce tumors or death in around 3%of the infected cows.Since Francis Peyton Rous's landmark study of identifying avian oncogenic virus,seven viruses have been found to cause human cancers worldwide.Recent findings by Buehring's group have suggested that BLV might be linked to human breast cancer.In a study of 218 women from four states in the USA,Buehring's group showed that the BLV positivity in women with breast cancer(67/114)is significantly higher than that in women without breast cancer(30/104).In this research,we made further study about the association between BLV and breast cancer with samples of patients with breast cancer from Anhui,Jiangsu and Shanghai in China.Part one:Establishment of a detection method of bovine leukemia virus by loop-mediated isothermal amplificationA variety of existing techniques are available for diagnosis of EBL worldwide,including histopathological test,hematological examination,the serological methods and molecular assay.The loop-mediated isothermal amplification(LAMP)method is a novel technique for the amplification of nucleic acid under isothermal conditions.In this study,according to reference of BLV genes released on GenBank,we designed six sets of primers for LAMP assay based on the six conserved region.With professional online software,we explored and optimized the conditions of LAMP assay,such as the best proportion of outer primers and inner primers,concentration of Mg2+,dosages of Bst 2.0 DNA polymerase and concentration of dNTPs.At last,specificity and sensitivity test were also carried out,and the results of LAMP assay were directly observed in color changes by adding calcein reagent.The LAMP assay for targeting the BLV region can detect at least 1 copy of proviral DNA and its sensitivity is equivalent to or greater than the conventional single PCR.In addition,amplification is obtained within 1 h by incubating a single tube in a water bath.The results of the LAMP assay showed a high level of agreement with FRET-qPCR.This suggests that the LAMP assay is a rapid,sensitive and simple method for the diagnosis of BLV infection,and is available for use in local laboratories without any special equipment.Part two:Study about association between bovine leukemia virus and breast cancer in Chinese patientsBreast cancer has raised great public concern,while the cause of breast cancer is still unknown.And the prevalence of BLV among cows in China is high.As more and more consumption of milk we have in our dailylife,the conjecture of association between BLV and human breast cancer may raise public concerns worldwide.For the high prevalence of BLV in Anhui,Jiangsu and Shanghai in prior study,all the samples of patients with breast cancer were all collected in these provinces in this study.We collected 91 samples of frozen mammary tissue from Shanghai(n=66)and Jiangsu(n=25).In addition,we collected 160 bloood samples of patients with breast cancer from Jiangsu(n=140)and Anhui(n=20),100 blood samples of healthy person from Jiangsu and 150 blood samples from cows in Anhui,Beijing,Heilongjiang and Jiangsu.In this study,we used several detection methods to study the association between BLV and breast cancer,including one PCR method we established before,five PCR methods in previous study reported the association between BLV and breast cancer,one LAMP method we established in this study and a commercial ELISA test kit based on gp51.FRET-qPCR and LAMP analysis revealed that BLV was positive in 50.0%of the cattle blood samples but was negative in the blood from women with or without breast cancer.ELISA revealed that 47.3%of the cattle blood samples were BLV antibody positive,whereas none of the blood samples from women with or without breast cancer were BLV antibody positive.Breast cancer has raised great public concern,while the cause of breast cancer is still unknown.And the prevalence of BLV among cows in China is high.As more and more consumption of milk we have in our dailylife,the conjecture of association between BLV and human breast cancer may raise public concerns worldwide.In conclusion,we established a rapid,sensitive loop-mediated isothermal amplification(LAMP)assay for diagnosis of BLV infection.Simultaneously,the unambiguous data in this study did not support the statement that BLV might be linked to human breast cancer.Further investigations are warranted to explore the association of BLV with breast cancer in patients with different genetic backgrounds and from different countries as well as their exposure to milk and the levels of infection in dairy herds.