1 INTRODUCTION

Since March 11, 2020, when the World Well being Group declared COVID-19 a pandemic, your entire globe has been scrambling for solutions concerning the virus SARS-CoV-2 (quick for extreme acute respiratory syndrome coronavirus 2). As of October 11, 2020, over 36 million instances of COVID-19 and 1 million deaths have been reported.1 A lot of the scientific analysis neighborhood’s efforts have been directed towards discovering the most affordable, quickest, and best option to detect a constructive case of COVID-19.2, 3 Efficient testing permits for contact tracing and applicable quarantining measures, which might curtail the unfold of the virus.4 Undergraduate biochemistry college students at Franklin Pierce College sought to carry out their very own COVID-19 assessments on environmental samples from the Rindge campus. This three-week laboratory module gives instruction in molecular instruments similar to real-time polymerase chain response (RT-PCR) in addition to the method by which a scientific diagnostic take a look at may be designed and executed.

This laboratory experiment follows the format of a course-based analysis experiences (CURE). Programs in biochemistry and molecular biology are enriched by course-based analysis experiences. The U.S. federal “Have interaction to Excel” report really helpful widespread adoption of energetic studying via problem-based studying similar to CURE laboratories5 (Gates, 2012). CUREs have been proven to extend studying,6 enhance persistence in science majors7, 8 in addition to having constructive impacts on appreciation for science and profession aspirations.9 Some analysis even helps the advantages of CUREs when the analysis objectives will not be met.10

RT-qPCR is a way whereby RNA is transformed to complementary DNA (cDNA) by reverse transcriptase enzyme, adopted by quantitative PCR (qPCR). As with normal PCR, qPCR includes amplification of DNA utilizing cycles of denaturation, annealing, and elongation. Nonetheless, with qPCR, the presence of a fluorescent dye that binds double-stranded DNA (dsDNA) permits for quantitation. The larger the quantity of RNA, the extra cDNA created and the less doubling cycles earlier than the fluorescence may be detected.11 Thus, a smaller variety of cycles to detect fluorescence, the extra RNA within the unique pattern. Quite a few articles have been printed that present undergraduate workouts utilizing qPCR or RT-PCR. Whereas a few of these sources use the acronym RT-PCR to face for real-time PCR,12, 13 others use RT-PCR to face for reverse transcription PCR14 and a few simply discuss with the method as qPCR.15 Some COVID testing protocols use the time period RT-qPCR,16 however our process shouldn’t be a very quantitative PCR (qPCR) as a result of we didn’t calculate beginning RNA calculations nor use a housekeeping gene for comparability of gene expression; thus, we’ll discuss with the approach as RT-PCR. In our experiment, college students investigated the presence of SARS-CoV-2 RNA on high-touch surfaces by swabbing them and performing RT-PCR on the samples.

RT-PCR has been the commonest diagnostic measure employed to detect the presence of SARS-CoV-2 on nasopharyngeal swabs.17, 18 To make use of this methodology as a diagnostic software for SARS-CoV-2 RNA, a fluorescence threshold (CT) is outlined. If the pattern reaches the fluorescence threshold in a small variety of PCR cycles (low CT), that pattern is constructive for SARS-CoV-2 RNA, because it signifies that there was a bigger quantity of RNA within the unique pattern. If CT could be very massive, or if it by no means reaches the fluorescence threshold, that pattern is detrimental for SARS-CoV-2 RNA. Correlations between illness development and CT values have been established,19 in addition to correlations with culturable virus.20, 21

This paper describes the method of how the undergraduate biochemistry class designed and ran their very own RT-PCR assessments to detect the potential presence of SARS-CoV-2 on campus. This lab was designed to reveal RT-PCR to the undergraduate biochemistry class, in addition to to doubtlessly detect samples that have been constructive for SARS-CoV-2 on campus. The artificial constructive management was based mostly on a process by Meza-Robles et al for one-step nested SARS-CoV-2 RT-PCR.22 This train makes use of no virus, so it includes no further security issues. Pedagogical benefits of the experiment embrace problem-based studying, experimental design, scientific purposes and a real-world utility.

2 PREREQUISITE KNOWLEDGE AND LEARNING OBJECTIVES

College students taking part on this train ought to have handed undergraduate common biology. They need to have a primary understanding of the central dogma of biology, transcriptional equipment and the method of polymerase chain response. The scholars needn’t have prior information or expertise with RT-PCR. Instructors ought to have some expertise with primary molecular methods and RT-PCR and entry to instrumentation.

ASBMB lists quite a few fascinating studying objectives related to understanding and manipulation of DNA and RNA, in addition to experimental design and information evaluation. These studying objectives are foundational ideas beneath the class of “Data Storage and Switch.”23 Our observable studying goals for this analysis module have been that the scholar will be capable to reveal understanding of the molecular biology, plan and design an experiment based mostly on major literature, develop a process that meets assigned objectives, execute their experimental plan in a collaborative style, and analyze and talk the outcomes.

3 TIMING

This lab was administered in a sequential style the place every lab group collected samples, however they collaborated vertically on the process and no teams accomplished all steps. This was partially necessitated by the necessity to socially distance within the laboratory sections as a result of COVID pandemic. Because of this, we had three to 4 lab classes per week and the venture lasted lower than 4 weeks. The timetable under displays the variety of 2-hour classes required to finish the lab as if one group had accomplished all of them sequentially. Different timing configurations are additionally potential: an teacher might have one group create the constructive management on the similar time that one other group ran the reverse transcription, or have a lab assistant put together the gel prematurely. Relying on class measurement, group measurement might vary from two to 4 college students.

4 weeks earlier than the lab started, we obtained the worth quote for the primers and started ordering. The three oligos for the constructive management have been the costliest (~$115 every). The week earlier than the lab started, informational supplies have been posted (Supporting Data). Notice that this requires college students to spend per week studying over the supplies and plan out their experiments. To orient the teams, prelab lectures at all times included a evaluation of the stream chart of your entire venture (Supporting Data). In actuality, a minority of scholars had rigorously learn the supplies and ready a full experimental protocol. Nonetheless, they have been in a position to ask questions and make clear their understanding throughout lab interval one because it was simply buffer preparation and sampling.

Lab 1—Throughout lab interval one, the scholars ready buffers, labeled tubes, distributed sterile swabs and every pupil swabbed a campus location. Lab 2—College students ready the templateless PCR to create the constructive management. Lab 3—College students took the thawed samples and ran the reverse transcription response. Lab 4—College students poured the agarose gel and made the buffers and ready the electrophoresis gel. Lab 5—College students loaded the gel with the ladder and constructive management, ran the gel, stained it with SYBRSafe and visualized it. Lab 6—College students arrange the RT-PCR, and ran the instrument.

The scholars got the expectation that their process and any findings have been posted to the shared digital lab pocket book inside 24 h in order that different teams might make the most of that data to proceed. This didn’t at all times happen; nonetheless, the lab report included a listing of the distribution of labor, and a peer analysis protocol to assist the teacher acknowledge college students’ contributions.

4 PROCEDURE

4.1 Supplies

Supplies wanted for the analysis module included synthesized oligonucleotides and primers, typical molecular biology reagents and tools and RT-qPCR instrumentation. College students weren’t given a lab process. As a substitute, for an inquiry lab, they have been supplied with background data and first literature (Supporting Data). A pattern pupil protocol is supplied (Supporting Data).

4.2 Sampling

The CDC Diagnostic Panel gives in depth steering on sampling, dealing with and preserving samples, primer alternative, reagents used, and process.24 We solely adopted their procedures loosely. College students might select a physiological buffer with tetracycline antibiotic. Swabs have been dipped in buffer, wiped twice over a floor, then damaged off into the gathering tube and capped. Collected samples have been saved at −80°C till use.

4.3 Era of the constructive management

An artificial, non-infectious sequence was generated to function a constructive management, based mostly on a paper by Meza-Robles et al.,22 utilizing templateless PCR to create a big constructive management sequence based mostly on the SARS-CoV2 ORF1ab gene sequence. Three oligonucleotides (1COV, 2COV, 3COV) have been synthesized (Desk S1) and assembled by templateless PCR. Templateless PCR depends on overlapping oligonucleotides in a PCR response with polymerase grasp combine to generate a contiguous sequence (Determine 1) that may be then detected by PCR. The sequence and biking circumstances have been as utilized in Meza-Robles22 after which, the product was re-amplified utilizing nested primers beneath the identical biking parameters. The ensuing fragment served as an artificial constructive management for the ORF1ab gene of SARS-CoV2 and was saved at −20°C. The unique product of the 1COV-2COV-3COV templateless PCR will also be used as a constructive management with the 1COFw and 1CORw primers, however the yield of the templateless PCR is usually low. Notice that Meza-Robles22 constructed their constructive management in a one-step nested PCR, however we carried out it in two steps, with no intermediate purification, for the sake of simplicity.

image
Cartoon illustrating templateless polymerase chain response (PCR). Three oligos have been synthesized (IDT) in accordance with Meza-Robles et al.22 with overlapping sequences. When joined by Taq polymerase, they assemble right into a contiguous strand in low effectivity, leading to a combined inhabitants of merchandise. End PCR is then carried out to pick out for the specified product utilizing primers (1COFw and 1CORw) complementary to the ends of the entire product. This amplifies the constructive management, on this case a partial sequence of SARS-CoV2 ORF1a

4.4 Gel electrophoresis

Electrophoresis of the constructive management was run on a 2.5% agarose gel with the constructive management generated by templateless PCR, detrimental management, and a ladder. The constructive management PCR product was purified from the gel slices utilizing a spin-column gel extraction equipment in accordance with producer’s directions.

4.5 RNA extraction

The intention was to have the scholars evaluate our RNA purification equipment to warmth extraction to launch the RNA from the virus. The scholars have been supplied with technical data for the RNA purification equipment and a journal article that reported the success of the warmth launch within the absence of RNA purification.16 Nonetheless, the primary group of scholars tried the warmth launch and it labored nicely, so all of them selected warmth extraction.

4.6 First-Strand synthesis and RT-PCR

Utilizing a bought equipment, first-strand synthesis (FSS) was accomplished to generate cDNA that would then be amplified in RT-PCR. Every environmental pattern was reverse transcribed along with one detrimental management per group that contained no enzyme. DNA generated from FSS and the constructive management have been run via RT-PCR. The thermocycler was set to run for 40 cycles with an annealing temperature of 53°C. A soften curve was carried out with SYBR-Inexperienced fluorescence information assortment after every 0.5°C enhance in temperature.

4.7 Security and waste disposal

Though the samples used have been environmental samples—and though some supplies, such because the constructive management and electrophoresis waste, will not be hazardous—all merchandise from the experiment have been collected as biohazard and autoclaved so as to eradicate the opportunity of COVID-positive environmental samples being disposed of inappropriately. Goggles, gloves, and fabric or surgical masks have been worn at some stage in the experiment. A HEPA air filter ran constantly and hand sanitizer was accessible.

5 RESULTS

The laboratory efficiently detected SARS-CoV-2 RNA, as judged by CT values (Desk 1). Each laboratory sections detected excessive concentrations of the constructive management (CT 1–2 cycles). Each sections had environmental samples that examined constructive for SARS-CoV-2 RNA, principally low ranges that have been unlikely to be infectious. One environmental pattern did have a CT worth of 23, which might be per an infectious viral load.19

TABLE 1.
RT-PCR outcomes
Properly Fluorophore Pattern CT Soften temperature (°C)
F01 SYBR None NaN None
F02 SYBR None NaN None
F03 SYBR + 1.008959 74.5
F04 SYBR None NaN None
F05 SYBR Pattern 3 23.10921 75.5
F06 SYBR NaN None
F07 SYBR NaN None
F08 SYBR None NaN None
F09 SYBR Pattern 2 31.38888 74
F10 SYBR Pattern 1 31.79547 75.5
  • Notice: RT-PCR Information. Information proven is from the part with probably the most constructive samples. Properly signifies arbitrary pattern place; Fluorophore SYBR is SybrGreen; Pattern-(+) signifies artificial constructive management; Pattern-(−) signifies—reverse transcriptase detrimental management or—template detrimental management. CT signifies the variety of cycles to achieve threshold detection; Soften temperature is the temperature for the dsDNA to denature.
  • Abbreviation: RT-PCR, real-time polymerase chain response.

The soften curves confirmed a drop in fluorescence at related temperatures, because the double-stranded PCR product strands separated (Determine S1A). Those self same information are additionally proven as a change in slope of the curve, leading to a peak on the “soften temperature” (Determine S1B). As a result of the soften temperature depends upon the guanine-cytosine content material (and distribution) of the product, overlapping peaks are per equivalent PCR merchandise.

6 CHALLENGES

We’ve got solely run this lab train as soon as, however we have been in a position to efficiently full the module with few difficulties. With many molecular biology lab workouts, the commonest difficulties come up from poor use of micropipettors or use of incorrect reagents. That is notably essential for PCR. Most of our biochemistry college students have had two earlier programs utilizing micropipettors, however we nonetheless have higher success after we require profitable completion of a lab module calibrating the micropipettors firstly of the semester. College students with earlier publicity to quantitative switch of small volumes and PCR ought to be capable to efficiently full the lab.

As was evident from the prelab quiz and lack of progress firstly of the interval, some college students didn’t put together in any respect earlier than the lab interval which, unsurprisingly, inhibited their means to course of the procedures. Virtually all the college students struggled to grasp the position and mechanism of the templateless PCR. The problem was compounded by the truth that some teams didn’t take part within the hands-on templateless PCR preparation. We suggest a person tutorial (Determine 1) or explanatory video on that approach alone (e.g., Reference 25) or, when potential, have not less than one group in every part carry out the templateless PCR.

Essentially the most placing problem college students encountered, although, is a ubiquitous one with undergraduate group tasks: a few of the college students didn’t contribute equally to the communication, writing, and revision course of. Two teams needed to be reminded to submit their information. As a result of college students have been posting of their digital notebooks individually, particular person grades mirrored their effort (roughly 5% penalty for the lab, the place notebooks have been price 30% of their course grade), but it surely created frustration for lab companions.

7 STUDENT WRITE-UP

Earlier than the primary lab, college students accomplished a prelab quiz. Our college students carried out a single RT-PCR run per lab part, in order that they have been required to put in writing up a single lab report per part. Our evaluation utilized grading of particular person lab notebooks (simply the stream chart and the part the scholar pair labored on) and a joint lab report for the lab part. The joint lab pocket book adopted the format of a typical temporary, formal lab report (Summary, Introduction, Strategies, Outcomes, Evaluation, Conclusions).

8 STUDENT SURVEY FEEDBACK

College students participated on this and one different analysis module within the class. An abbreviated model of the CURE survey9, 26 was administered earlier than (N = 12) and after (N = 20) the scholars accomplished the analysis modules. Between the pre-course survey and the post-course survey, there have been only some variables that shifted. The scholars apparently had a superb sense within the pre-survey that actual science can proceed in a non-linear style however few had participated in a course the place the scholars had enter into the experiment design (“little” or “no” expertise 83%).

Following the analysis modules, pupil suggestions was constructive however not uniformly so. 80% of the scholars (N = 20) disliked doing the lab sequentially or writing a single lab report as a bunch. 85% of the scholars mentioned that the tasks didn’t have an effect on their attitudes or plans about graduate examine however 10% mentioned that they have been considerably extra more likely to take into account graduate schooling after doing the analysis expertise and one (5%) mentioned that it made them much less more likely to take into account postgraduate analysis. 55% mentioned it gave them a greater sense of how scientists take into consideration experiments and plan them out, whereas 45% mentioned that the side that they realized probably the most from was analyzing unique information.

Requested in an open-ended query what they most loved within the analysis expertise, one pupil famous that they tremendously most popular the fundamental labs on spectrophotometry to the analysis expertise as a result of they felt extra assured of their understanding and talent to finish the work. Nonetheless, widespread responses have been that they favored the flexibleness in scheduling (20%), studying new methods (20%), studying how persons are doing COVID testing “in the true world” (10%). One pupil famous that they felt that they have been “…in a position to expertise true scientific discovery in an expert lab setting.”

9 EXTENSION ACTIVITIES

A spread of potentialities exist for extension actions. We mentioned experimental design and the way COVID testing was being carried out with our courses. However, a full exploration of sensitivity, specificity, sampling methods and scientific variation might be included.

College students might rigorously evaluate the warmth extraction versus conventional RNA extraction utilizing chloroform/isopropanol or a spin column equipment. Utilizing a single label (SYBR inexperienced) implies that just one goal sequence may be assayed without delay as a result of the fluorescent dye will bind to any dsDNA that’s amplified; use of multiple fluorophore would allow multiplex RT-PCR for multiple goal in a single tube.11 Alternatively, probes may be designed which have their very own sequence specificity, reducing the possibility of non-specific binding, notably for eukaryotic transcriptomes.27

Constructive controls might be generated for different genes (Supporting Data). The qPCR may be accomplished quantitatively, as demonstrated by Hancock et al.13 or McCauslin et al.15 This may require higher quantitation of the constructive management and will embrace a spike-in of a small quantity of the constructive management in pattern duplicates to rule out interfering substances and confirm quantitation. In future years, SARS-CoV-2 will likely be much less of a well being concern, however these strategies might be simply tailored to coronaviruses similar to HCoV-OC43 that trigger the widespread chilly,28 or different delicate viral pathogens by merely altering the sequences for the constructive management and for the primers.

10 CONCLUSIONS

This analysis module gives upper-level undergraduates the chance to design and safely conduct an experiment that mixes molecular biology design with a real-world public well being utility. Experimental procedures which were beforehand encountered similar to agarose gel electrophoresis are strengthened. The train additionally requires the scholars to check printed experimental procedures and incorporate them into their very own design.

The flexibility to work with DNA and RNA is key for all biologists and biochemists. Using RT-qPCR is a delicate approach that’s handy for comparative gene expression. Though RNA-seq is changing into extra widespread and extra reasonably priced, RT-qPCR continues to be extensively utilized. RT-PCR additionally has purposes in assessing DNA yield and in forensic science.12

Templateless PCR, used to generate the constructive management, is a way whereby massive oligos may be designed with overlapping terminal sequences and assembled right into a contiguous single-stranded product by PCR. The approach is essential for artificial biology and has been used with undergraduates to generate massive partially artificial chromosomal segments.29

The scholars had been warned that we’d don’t have any constructive environmental samples and that any constructive samples would solely point out RNA, not essentially infectious virus, and never essentially point out that there had been sufficient viral load to be infectious. We did, nonetheless, have a pattern that had excessive RNA ranges per infectious virus.19 Our findings have been nearly 2 weeks after sampling in areas that had common antiviral cleansing, however workers ensured further decontamination of the world. As well as, to keep away from any rumors or issues from college students, administration, or dad and mom, we additionally created a FAQ about our experiment and findings (Supporting Data).

The COVID-19 take a look at improvement module proved to be an efficient educating software for contemporary molecular biology methods. The subject was of extraordinarily excessive curiosity for the scholars and their enthusiasm was palpable. Primary abilities strengthened embrace viral physiology, transcription and reverse transcription, and experimental design. Matters that have been novel to the scholars included viral sampling and storage, templateless PCR, and use of qPCR instrumentation. This RT-PCR lab module supplied a real analysis expertise with present scientific purposes of crucial public well being significance.

ACKNOWLEDGMENTS

Assist was supplied by Franklin Pierce College Faculty of Well being and Pure Sciences. Ordering and lab administration was assisted by Lisa Bomba, Victoria Wellenstein, and Justin Haynes. The BioRad CFX 96-well qPCR instrument was funded by NH-INBRE (New Hampshire-INBRE via an Institutional Growth Award [IDeA], P20GM103506, from the Nationwide Institute of Basic Medical Sciences of the NIH). Figures have been generated in Abobe Illustrator. Because of the 2020 FPU Biochemistry class. The coed surveys have been administered beneath the approval of Franklin Pierce College IRB #SP11102020.

REFERENCES