Highlights
- •Rapid presumptive tests for saliva and vaginal secretion were developed using RT-RPA-LFD assay with STATH and SPINK5 markers.
- •The experiment could be accomplished within 10–15 min within the range of 30–42 ℃, excluding RNA extraction.
- •These assays have high specificity and sensitivity: 100 fg or 50 fg of saliva and vaginal secretion samples could be successfully detected.
Abstract
Identifying the origin of body fluids is a critical step in a forensic investigation.
One widely used method to identify human body fluids is based on the color visualization
of immune antigen detection strips for detecting hemoglobin in blood and prostate-specific
antigen in semen. It is highly imperative to construct an easy-to-perform, mRNA-based
method for the point-of-care identification of other human body fluids, such as saliva
and vaginal secretion. Here, we established specific strips with the mRNA markers
STATH (for saliva) and SPINK5 (for vaginal secretion) via reverse transcription recombinase
polymerase amplification (RT-RPA) and lateral flow dipstick (LFD) assays (RT-RPA-LFD).
RT-RPA could be accomplished in a single tube at a wide temperature range of 30–42 ℃
within 10–25 min if we do not count time for RNA extraction. The diluted RPA products
were added onto the LFD strip pad to visually observe the color change of the Control/Test
line. The tissue specificity and detection limit of the assays were evaluated using
the optimized reaction conditions of RPA at 37 ℃ for 15 min. The positive signals
of STATH were observed both in saliva and nasal secretions. SPINK5 was positive in
a template-dependent manner in 4 out of 30 female urine samples in addition to vaginal
secretion and menstrual blood samples. Cross-reactions were not detected in semen,
skin swabs, sweat, or male urine. Both assays were capable of detecting aged samples,
which were stored for 180 days (saliva) or 300 days (vaginal secretion) at room temperature.
Moreover, saliva or vaginal secretion was successfully detected in all kinds of mixtures
made from various body fluids. Overall, the rapid strip test method by the RT-RPA-LFD
assay is simple, time-saving and highly sensitive for estimating the tissue origin
of saliva and vaginal secretion. This method for the rapid RNA-based presumptive tests
of the tissue type of body fluids is easy to perform prior to a multiplex mRNA analysis,
which can demonstrate more reliable saliva or vaginal secretion identification.
Keywords
Abbreviations:
RT (Reverse transcription), RT-RPA (Reverse transcription recombinase polymerase amplification), LFD (Lateral flow dipstick), STR (Short tandem repeat), LAMP (Loop-mediated isothermal amplification)To read this article in full you will need to make a payment
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Article info
Publication history
Published online: October 06, 2022
Accepted:
October 5,
2022
Received in revised form:
September 25,
2022
Received:
June 23,
2022
Identification
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