The detection and identification of saliva in forensic samples by RT-LAMP
We report on a novel method for saliva identification by reverse transcription-loop-mediated isothermal amplification (RT-LAMP). In our previous report, real-time RT-LAMP was used for blood identification by using HBB detection as a model but in this advanced study, this method was refined for the identification of the more challenging body fluid of saliva. Expression of the18S rRNA gene was used as the internal control and the Statherin (STATH) gene as the saliva-specific marker. A turbidimeter was used for real-time detection of the RT-LAMP products, and confirmation was obtained that the real products were generated using: agarose gel electrophoresis, calcein fluorescence detection and/or enzymatic digestion. The specificity of the test was performed using 42 samples including 7 different body fluids, and the expression of STATH was only observed in all the saliva samples (6) with a threshold time of 39.4 ± 2.9 min. Sensitivity testing showed that RT-LAMP products for STATH were stably detected when the RNA template was not less than 6.25 ng. When the primer concentrations for STATH were two times that of 18S rRNA, saliva could be identified in the body fluid mixtures even at a ratio (saliva:semen) of 1:3 (without loop primer)/1:5 (with loop primer). A multiplex RT-LAMP was established to simultaneously amplify the 18S rRNA and STATH genes, and applied to the identification of saliva on ten non-probative cigarette butts. A positive result for saliva was obtained from all ten butts, even for those that returned a negative or ambiguous result using the amylase test. A direct RT-LAMP test is also reported where the RNA extraction step was omitted to speed the collection of data and all tests using either the simplex or multiplex RT-LAMP resulted in a positive response if saliva was present. Our data provide a simple and effective means to detect the presence of saliva.
KeywordsSaliva identification RT-LAMP Statherin (STATH) Multiplex RT-LAMP Non-probative evidence samples
This study was supported by the Ministry of Science and Technology in Taiwan (grant number NSC 101-2320-B-015-001 and MOST 104-2320-B-015 -001 -MY2).
This study was funded by the Ministry of Science and Technology in Taiwan (grant numbers NSC 101–2320-B-015-001 and MOST 104–2320-B-015-001 -MY2).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The human body fluids used in this study were collected from the volunteers using procedures approved by Antai Medical Care Cooperation Antai- Tian-Sheng Memorial Hospital Institutional Review Board in Taiwan (TSMH IRB No./ Protocol No.: 14–085-B1).
Informed consent was obtained from all individual participants included in the study.
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