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. 2022 Feb 28:13:837945.
doi: 10.3389/fpls.2022.837945. eCollection 2022.

Development and Validation of a Novel and Fast Detection Method for Cannabis sativa: A 19-Plex Short Tandem Repeat Typing System

Ruocheng Xia  1 , Ruiyang Tao  1 , Yiling Qu  1 , Xiaochun Zhang  1 , Huan Yu  2 , Chunyan Yuan  1   3 , Suhua Zhang  1 , Chengtao Li  1
Affiliations

Development and Validation of a Novel and Fast Detection Method for Cannabis sativa: A 19-Plex Short Tandem Repeat Typing System

Ruocheng Xia et al. Front Plant Sci. .

Abstract

In recent years, influenced by the legalization of Cannabis sativa in some countries and regions, the number of people who smoke or abuse C. sativa has continuously grown, cases of transnational C. sativa trafficking have also been increasing. Therefore, fast and accurate identification and source tracking of C. sativa have become urgent social needs. In this study, we developed a new 19-plex short tandem repeats (STRs) typing system for C. sativa, which includes 15 autosomal STRs (D02-CANN1, C11-CANN1, 4910, B01-CANN1, E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159, nH09, CS1, ANUCS 305, 3735, and ANUCS 302 and 9043), two X-chromosome STRs (ANUCS 501 and 1528), one sex-determining marker (DM016, on Y-chromosome), and a quality control marker (DM029, on autosome). The whole polymerase chain reaction (PCR) process could finish within 1 h, making the system suitable for fast detection. The PCR products were detected and separated with an Applied Biosystems 3500XL Genetic Analyser. Developmental validation studies indicated that the 19-plex typing system was accurate, reliable and sensitive, which could also deconvolute mixed C. sativa samples. Specifically, the sensitivity study showed that a full genotyping profile was obtainable with as low as 125 pg of C. sativa DNA. The species specificity study demonstrated that this multiplex has no cross-reactivity with common non C. sativa DNA. In the population study, a total of 162 alleles at 15 autosomal STRs and 14 alleles at two X-chromosome STRs were detected among 85 samples. The efficiency parameters, including the total discrimination power (TDP) and the combined power of exclusion (CPE) of the system, were calculated to exceed 0.999 999 999 999 988 and 0.998 455 889 684 078, respectively, further proving that the system could meet the needs of individual identification. To the extent of the known studies, this is the first study that included the C. sativa sex-determining marker. In conclusion, the developed new 19-plex STR typing system can successfully achieve the purposes of species identification, gender determination, and individual identification, which could be a powerful tool in tracing trade routes of particular drug syndicates or dealers or in linking certain C. sativa to a crime scene.

Keywords: Cannabis sativa; capillary electrophoresis (CE); developmental validation; multiplex system; polymerase chain reaction (PCR); short tandem repeats (STRs).

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Electropherogram of positive control DNA with the Cannabis sativa 19-plex typing system.
FIGURE 2
FIGURE 2
Electropherogram of the allelic ladder designed for the Cannabis sativa 19-plex typing system.
FIGURE 3
FIGURE 3
Sizing precision testing across 24 injections of the allelic ladder of the Cannabis sativa 19-plex typing system performed on the ABI 3500xl Genetic Analyser.
FIGURE 4
FIGURE 4
Sizing accuracy study of the Cannabis sativa 19-plex typing system performed on the ABI 3500xl Genetic Analyser. These data represent a total of 2,705 alleles from 100 samples.
FIGURE 5
FIGURE 5
Sensitivity testing of template DNA ranging from 15.625 pg to 2 ng. The average percentage of loci detected was against the DNA input quantity. The left Y-axis represents the average percentage of loci detected, and the right Y-axis represents the average peak height. Error bars show the SDs between three replicates.
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