title: Ultrasensitive Detection of Bacteria by Targeting Abundant Transcripts.
abstract: Molecular detection assays are increasingly becoming routine diagnostic techniques for bacterial infection; however, their sensitivities are restricted by the low concentrations of bacteria in clinical samples. Here, we report a new paradigm for ultrasensitive detection of bacteria. The principle of this approach is that by choosing highly transcribed genes as signature sequences and detecting both DNA and its RNA transcripts, assay sensitivity can be greatly improved. First, signature genes with abundant transcripts were screened by RNA-Seq. We confirmed that RT-PCR efficiently amplifies both DNA and RNA, while PCR amplifies only DNA. Unexpectedly, we found that the RNA extraction efficiency is relatively low, while simplified denaturation was more appropriate for transcript detection. For highly transcribed genes, RT-PCR consistently generated lower cycle threshold (Ct) values than those of PCR. The sensitivity of RT-PCR targeting abundant transcripts could detect quantities as low as one bacterium, which was not possible using PCR. Amplification of different genes among several other common bacteria also confirmed that transcript detection by RT-PCR is more sensitive than is DNA detection by PCR. Therefore, abundant transcript detection represents a universal strategy for ultrasensitive detection of bacteria.
authors: Institute of Zoonosis, Jilin University, 130062, Changchun, China.|Institute of Disease Control and Prevention, Academy of Military Medical Sciences, 100071, Beijing, China.
authors: Xinhui
authors: X
authors: Wang
authors: Institute of Zoonosis, Jilin University, 130062, Changchun, China.|Institute of Disease Control and Prevention, Academy of Military Medical Sciences, 100071, Beijing, China.
authors: Xinran
authors: X
authors: Li
authors: Wangjing Hospital, Academy of Traditional Chinese Medicine, 100102, Beijing, China.
authors: Shiwei
authors: S
authors: Liu
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, 100071, Beijing, China.
authors: Hang
authors: H
authors: Ren
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, 100071, Beijing, China.
authors: Mingjuan
authors: M
authors: Yang
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, 100071, Beijing, China.
authors: Yuehua
authors: Y
authors: Ke
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, 100071, Beijing, China.
authors: Liuyu
authors: L
authors: Huang
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, 100071, Beijing, China.
authors: Chao
authors: C
authors: Liu
authors: Institute of Zoonosis, Jilin University, 130062, Changchun, China.
authors: Bo
authors: B
authors: Liu
authors: Institute of Zoonosis, Jilin University, 130062, Changchun, China.|School of Medicine, Shihezi University, 832003, Shihezi, China.
authors: Zeliang
authors: Z
authors: Chen
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D002002:Brucella / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D017423:Sequence Analysis, RNA
chemical_list: D004269:DNA, Bacterial; D012329:RNA, Bacterial