Objective: To establish a single quantitative real-time PCR for the co-detection of human herpesvirus 6(HHV-6) and human herpesvirus 7(HHV-7) nucleic acids in clinical samples and cell matrix samples. Methods: The genomic nucleic acid sequences of representative HHV-6 and HHV-7 strains were analyzed. The homogenous sequenceswere selected to design the single probe and individual primers for HHV-6 and HHV-7. Through experimental comparison and analysis,it was determined that the use of mixed primers had no impact on detection. The Real-time PCR was established after the amplification temperature was optimized. The specificity,limit of detection(LOD) ,Limit of quantitation(LOQ) and linearity(accuracy) of Real-time PCR were validated. The method was used to detect the clinical samples and the cell matrix samples for biological products. The results were compared with those of nested PCR. Results: By comparison,the mixing of HHV-6 and HHV-7 primers had no effect on the detection performance,and HHV-6/HHV-7 could be detected separately or simultaneously. In the specificity test,the genomic DNA of 293 cells showed no interfere,and there were no amplification in samples containing PRV、VSV、VZV、EB1、EB2、EB3、CMV1/2/3、HSVI、HSVII. LOD and LOQ of the method are 1×102 copies and 1×103 copies,respectively. The linear range of the method is 1×103-1×107 copies. Real-time PCR and nested PCR were used to detect 86 clinical plasma samples and 15 biological production cell matrix samples. 8 samples and 4 samples of clinical samples were detected positive,respectively. And 15 cell matrix samples showed all negative results. Resultsof χ2 analysis showed that the detection sensitivity of Real-time PCR was significantly higher than nested PCR. Conclusion: The established real-time PCR can be used to detect HHV-6 and HHV-7 simultaneously in a single PCR reaction. The sensitivity of Real-time PCR is higher than the nested PCR,and it can be used to detect HHV-6 and HHV-7 in clinical plasma samples and cell matrix samples.
ZHANG Feng, GAO Li-ying, SONG Xue, YUAN Rong-rong, WU Xue-ling, FAN Jin-ping, MENG Shu-fang
. Establishment and application of quantitative real-time PCR for co-detection of HHV-6 and HHV-7[J]. Chinese Journal of Pharmaceutical Analysis, 2021
, 41(9)
: 1565
-1575
.
DOI: 10.16155/j.0254-1793.2021.09.09
[1] BARONE SR,KAPLAN MH,KRILOV LR.Human herpesvirus-6 infection in children with first febrile seizures[J]. J Pediatr,1995, 127(1):95
[2] ABLASHI DV,BERNEMAN ZN,KRAMARSKY B,et al.Human herpesvirus-7 (HHV-7):current status[J]. Clin Diagn Virol, 1995,4(1):1
[3] DANIEL KB,GERALDINA D,PHILIPE P.Human herpesvirus 6[J]. Clin Microbiol Rev,1997,10(3):521
[4] LINDA SW,WILLIAM JR,BALACHANDRAN N,et al.Human herpesvirus 7:antigenic properties and prevalence in children and adults[J]. J Virol,1991,65(11):6260
[5] DRAGO F,REBORA A.The new herpesviruses:emerging pathogens of dermatological interest[J]. Arch Dermatol,1999,135(1):71
[6] PARRA M,ALCALA A,AMOROS C,et al.Encephalitis associated with human herpesvirus-7 infection in an immunocompetent adult[J]. Virol J,2017,14(1):97
[7] REDDY S,ELIASSEN E,KRUEGER GR,et al.Human herpesvirus 6-induced inflammatory cardiomyopathy in immunocompetent children[J]. Ann Pediatr Cardiol,2017,10(3):259
[8] TOMASZEWSKA A,KRYSKO A,DZIECIATKOWSKI T,et al.Co-infections with cytomegalovirus and human herpesvirus type 7 in adult Polish allogeneic haematopoietic stem cell transplant recipients[J]. Arch Immunol Ther Exp (Warsz),2014,62(1): 77
[9] HILL JA,KOO S,GUZMAN SBB,et al.Cord-blood hematopoietic stem cell transplant confers an increased risk for human herpesvirus-6-associated acute limbic encephalitis:a cohort analysis[J]. Biol Blood Marrow Transplant,2012,18(11):1638
[10] LUKA J,OKANO M,THIELE G.Isolation of human herpesvirus-6 from clinical specimens using human fibroblast cultures[J]. J Clin Lab Anal,1990,4(6):483
[11] CHEN M,POPESCU N,WOODWORTH C,et al.Human herpesvirus 6 infects cervical epithelial cells and transactivates human papillomavirus gene expression[J]. J Virol,1994,68(2):1173
[12] CERMELLI C,CONCARI M,CARUBBI F,et al.Growth of human
herpesvirus 6 in HEPG2 cells[J]. Virus Res,1996,45(2):75
[13] OLIVEIRA SA,TURNER DJ,KNOWLES W,et al.Primary human herpesvirus-6 and -7 infections,often coinciding,misdiagnosed as measles in children from a tropical region of Brazil[J]. Epidemiol Infect,2003,131(2):873
[14] COHEN JI,FAHLE G,KEMP MA,et al.Human herpesvirus 6-A,6-B, and 7 in vitreous fluid samples[J]. J Med Virol,2010,82(6):996
[15] SADA E,YASUKAWA M,ITO C,et al.Detection of human herpesvirus 6 and human herpesvirus 7 in the submandibular gland, parotid gland,and lip salivary gland by PCR[J]. J Clin Microbiol, 1996,34(9):2320
[16] PELLETT MR,HAND J.Human herpesvirus 6,7,and 8 in solid organ transplantation:Guidelines from the american society of transplantation infectious diseases community of practice[J]. Clin Transplant,2019,33(9):e13518
