{"title":"用于法医体液鉴定的多路实时定量PCR分析的部分验证","authors":"Courtney Lynch , Rachel Fleming","doi":"10.1016/j.scijus.2023.10.004","DOIUrl":null,"url":null,"abstract":"<div><p>Confirmatory body fluid identification using messenger RNA (mRNA) is a well-established technique to address issues encountered with conventional testing – such as poor sensitivity, specificity, and a lack of available tests for all body fluids of interest. For over a decade, endpoint reverse-transcription polymerase chain reaction (RT-PCR) assays have been used in forensic casework for such purposes. However, in comparison with real-time quantitative RT-PCR (RT-qPCR), endpoint RT-PCR has lower sensitivity, precision, and linear dynamic range.</p><p>This research details the multiplexing and partial validation of confirmatory RT-qPCR assays. We have previously described novel assays for a range of body fluid targets and identified an optimal commercial kit for their amplification. Here, multiplexing was undertaken to form three assays: circulatory blood (<em>SLC4A1</em>) and menstrual fluid (<em>STC1</em>), saliva (<em>HTN3</em>) and vaginal material (<em>CYP2B7P</em>), and spermatozoa (<em>PRM1</em>) and seminal fluid (<em>KLK2</em>), all including a synthetic internal control RNA.</p><p>Partial validation of the multiplexed assays incorporated the MIQE guidelines, ISO requirements, and SWGDAM guidelines. Using receiver operating characteristic (ROC) curves, each marker was significantly different from an uninformative assay and optimal cut-offs were all above 35 cycles. All assays showed a wide LDR (ranging from 3 to 5 logs with most R<sup>2</sup> > 0.99), and high precision (most mean CV < 1 %). <em>STC1</em> showed some instances of sporadic expression in blood, semen, and vaginal material at high C<sub>T</sub> values. <em>CYP2B7P</em> showed off-target expression in semen and blood. The sensitivities were approximated as; saliva: 1 in 1,000 dilution of a whole buccal swab, circulatory blood: 0.01–0.1 µL blood, menstrual fluid: 1 in 10,000 dilution of a whole menstrual swab, spermatozoa: 0.001 µL semen, seminal fluid: 0.01 µL semen, and vaginal material: 1 in 1,000 dilution of a whole vaginal swab.</p><p>A total of 16 mock body fluid extract mixtures and 18 swab mixtures were tested and had 100% and 99% detection of target markers below each specific cut-off, respectively. Some mixtures containing high volumes of blood and semen showed off-target <em>CYP2B7P</em> expression. The successful application of a probabilistic model to the RT-qPCR data was also demonstrated. Further work will involve full developmental validation.</p></div>","PeriodicalId":49565,"journal":{"name":"Science & Justice","volume":null,"pages":null},"PeriodicalIF":1.9000,"publicationDate":"2023-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":"{\"title\":\"Partial validation of multiplexed real-time quantitative PCR assays for forensic body fluid identification\",\"authors\":\"Courtney Lynch , Rachel Fleming\",\"doi\":\"10.1016/j.scijus.2023.10.004\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Confirmatory body fluid identification using messenger RNA (mRNA) is a well-established technique to address issues encountered with conventional testing – such as poor sensitivity, specificity, and a lack of available tests for all body fluids of interest. For over a decade, endpoint reverse-transcription polymerase chain reaction (RT-PCR) assays have been used in forensic casework for such purposes. However, in comparison with real-time quantitative RT-PCR (RT-qPCR), endpoint RT-PCR has lower sensitivity, precision, and linear dynamic range.</p><p>This research details the multiplexing and partial validation of confirmatory RT-qPCR assays. We have previously described novel assays for a range of body fluid targets and identified an optimal commercial kit for their amplification. Here, multiplexing was undertaken to form three assays: circulatory blood (<em>SLC4A1</em>) and menstrual fluid (<em>STC1</em>), saliva (<em>HTN3</em>) and vaginal material (<em>CYP2B7P</em>), and spermatozoa (<em>PRM1</em>) and seminal fluid (<em>KLK2</em>), all including a synthetic internal control RNA.</p><p>Partial validation of the multiplexed assays incorporated the MIQE guidelines, ISO requirements, and SWGDAM guidelines. Using receiver operating characteristic (ROC) curves, each marker was significantly different from an uninformative assay and optimal cut-offs were all above 35 cycles. All assays showed a wide LDR (ranging from 3 to 5 logs with most R<sup>2</sup> > 0.99), and high precision (most mean CV < 1 %). <em>STC1</em> showed some instances of sporadic expression in blood, semen, and vaginal material at high C<sub>T</sub> values. <em>CYP2B7P</em> showed off-target expression in semen and blood. The sensitivities were approximated as; saliva: 1 in 1,000 dilution of a whole buccal swab, circulatory blood: 0.01–0.1 µL blood, menstrual fluid: 1 in 10,000 dilution of a whole menstrual swab, spermatozoa: 0.001 µL semen, seminal fluid: 0.01 µL semen, and vaginal material: 1 in 1,000 dilution of a whole vaginal swab.</p><p>A total of 16 mock body fluid extract mixtures and 18 swab mixtures were tested and had 100% and 99% detection of target markers below each specific cut-off, respectively. Some mixtures containing high volumes of blood and semen showed off-target <em>CYP2B7P</em> expression. The successful application of a probabilistic model to the RT-qPCR data was also demonstrated. Further work will involve full developmental validation.</p></div>\",\"PeriodicalId\":49565,\"journal\":{\"name\":\"Science & Justice\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.9000,\"publicationDate\":\"2023-10-16\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Science & Justice\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1355030623001041\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"MEDICINE, LEGAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Science & Justice","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1355030623001041","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MEDICINE, LEGAL","Score":null,"Total":0}
Partial validation of multiplexed real-time quantitative PCR assays for forensic body fluid identification
Confirmatory body fluid identification using messenger RNA (mRNA) is a well-established technique to address issues encountered with conventional testing – such as poor sensitivity, specificity, and a lack of available tests for all body fluids of interest. For over a decade, endpoint reverse-transcription polymerase chain reaction (RT-PCR) assays have been used in forensic casework for such purposes. However, in comparison with real-time quantitative RT-PCR (RT-qPCR), endpoint RT-PCR has lower sensitivity, precision, and linear dynamic range.
This research details the multiplexing and partial validation of confirmatory RT-qPCR assays. We have previously described novel assays for a range of body fluid targets and identified an optimal commercial kit for their amplification. Here, multiplexing was undertaken to form three assays: circulatory blood (SLC4A1) and menstrual fluid (STC1), saliva (HTN3) and vaginal material (CYP2B7P), and spermatozoa (PRM1) and seminal fluid (KLK2), all including a synthetic internal control RNA.
Partial validation of the multiplexed assays incorporated the MIQE guidelines, ISO requirements, and SWGDAM guidelines. Using receiver operating characteristic (ROC) curves, each marker was significantly different from an uninformative assay and optimal cut-offs were all above 35 cycles. All assays showed a wide LDR (ranging from 3 to 5 logs with most R2 > 0.99), and high precision (most mean CV < 1 %). STC1 showed some instances of sporadic expression in blood, semen, and vaginal material at high CT values. CYP2B7P showed off-target expression in semen and blood. The sensitivities were approximated as; saliva: 1 in 1,000 dilution of a whole buccal swab, circulatory blood: 0.01–0.1 µL blood, menstrual fluid: 1 in 10,000 dilution of a whole menstrual swab, spermatozoa: 0.001 µL semen, seminal fluid: 0.01 µL semen, and vaginal material: 1 in 1,000 dilution of a whole vaginal swab.
A total of 16 mock body fluid extract mixtures and 18 swab mixtures were tested and had 100% and 99% detection of target markers below each specific cut-off, respectively. Some mixtures containing high volumes of blood and semen showed off-target CYP2B7P expression. The successful application of a probabilistic model to the RT-qPCR data was also demonstrated. Further work will involve full developmental validation.
期刊介绍:
Science & Justice provides a forum to promote communication and publication of original articles, reviews and correspondence on subjects that spark debates within the Forensic Science Community and the criminal justice sector. The journal provides a medium whereby all aspects of applying science to legal proceedings can be debated and progressed. Science & Justice is published six times a year, and will be of interest primarily to practising forensic scientists and their colleagues in related fields. It is chiefly concerned with the publication of formal scientific papers, in keeping with its international learned status, but will not accept any article describing experimentation on animals which does not meet strict ethical standards.
Promote communication and informed debate within the Forensic Science Community and the criminal justice sector.
To promote the publication of learned and original research findings from all areas of the forensic sciences and by so doing to advance the profession.
To promote the publication of case based material by way of case reviews.
To promote the publication of conference proceedings which are of interest to the forensic science community.
To provide a medium whereby all aspects of applying science to legal proceedings can be debated and progressed.
To appeal to all those with an interest in the forensic sciences.