The study design considers several factors that may affect DNA recovery from heroin samples: 1 purity of heroin, 2 quantity of sample grams , 3 extraction method, and 4 type of heroin sample white or brown powder or black tar. Heroin samples with sufficiently high quantities total mass were selected to enable consistent comparisons across extraction methods Table 2. The input mass of heroin samples were evaluated to determine the minimum mass required for significant DNA yield. The samples consisted of a white powder heroin-HCl Each extraction method was evaluated using approximately 3.
The remaining samples did not have the sufficient quantity of material to perform this evaluation. Note, individual heroin samples were limiting and many times did not have the sample mass to permit many replicates for comparison. This limited our ability to compare the extraction methods and unequivocally evaluate the DNA extraction methods and conditions. Note, no picture of WP-1 is available.
Like-samples were then pooled during post-extraction concentration. The sample that was not lysed appeared darker and had insoluble components present that could not be removed via centrifugation. The lysed sample went into solution rapidly and had no visible insoluble components. The white powder heroin appeared to go into solution faster than the other samples and consistently had fewer insoluble components removed during the initial preparation of samples for extraction.
This step precipitates proteins, large chromosomal DNA molecules, and other containments and will promote DNA adsorption to the silica membrane. Appearance of white powder heroin samples prepared with the lysis left and without lysis right using the Maxwell 16 RNA LEV extraction method. This indirectly provides a relative assessment of the starting quantity of heroin and poppy DNA in the sample and will allow preliminary comparison of DNA quantity and the heroin purity and type.
The 1. In practice, we recommend the use of 2. However, for the purposes of this study, 1. The NTC samples across runs had reproducible signals, with melting temperatures between This supports the conclusion that the Maxwell extraction method is not the appropriate method for samples of this type.
The results from the first run confirmed the successful amplification of poppy DNA from heroin without any background noise from the tRNA Fig. The no-template control, yeast tRNA, and one replicate of the reagent blank, did not show detectable activity in the amplification plot.
The first replicate of the reagent blank had a C p value of The heroin samples had C p values of approximately The amplification displays the accumulation of double stranded DNA vs cycle, thus the increase in fluorescence at lower cycles indicate higher concentrations of DNA. A comparison of commercially available illustra GenomiPhi v3 and in-house whole-genome amplification methods was performed. We hypothesized that the presence of damaged DNA lesions, deamination may hinder the DNA polymerase used in the commercial kit.
The in-house method included use of commercially available random hexamers DNA fragments of six bases and the 2-step amplification. The study was restricted to two samples due to the limited availability of samples.
The results not shown indicated that in the current state, neither method improved the quantity of amplifiable DNA. The data further demonstrated that both black tar and powder heroin can be amplified at low levels Table 5 and Fig. Black tar is crudely manufactured and therefore the dense, sticky, and gummy sample matrices vary from sample to sample.
Despite this challenging matrix, we were able to successfully extract Papaver somniferum L. DNA from black tar heroin. Note the table in the upper left containing the cycle threshold values Cp. Raw opium is produced through the collection and, typically, sun drying of poppy latex.
This minimal processing leads to the retention of cellular material and maintains the integrity of the DNA. The challenge for raw opium is in the purification of DNA from inhibiting compounds and the texture of the sample. The first experimental objective was to identify the lowest raw opium sample mass that would provide reliable and robust results. The absorbance data obtained from the spectrophotometer provides an indication of sample purity, and thus indicates potential PCR performance.
The results indicate that the 0. The target mass may vary due to the multitude of different characteristics displayed in an illicit opium sample, e. The differences in quality of the material dried, gummy or cooked will likely play the most significant role in determining the amount of starting material Supplementary Information: Fig.
A sample that is overly desiccated and exposed to high temperatures cooked will undoubtedly require an increase in the amount of starting material. The second study focused on identifying an efficient DNA extraction method that results in an acceptable yield and purity of DNA. The Qiagen-based method will likely result in an increasingly pure extract when compared to the Promega method, with values of 1.
The average ratio for the Qiagen-extracted samples improved to 1. The higher moisture content of the opium samples had a negative impact on the quality of results. To mitigate this problem we attempted two approaches: pretreatment with liquid nitrogen and air-drying. The preferred sample pre-processing method included air-drying in a fume hood and, as needed, treatment of the sample with liquid nitrogen followed by physical disruption with a mortar and pestle and bead-beating.
This greatly increased the surface area of the opium sample exposed to the lysis buffers while simultaneously decreasing the activity of any endogenous DNA degrading DNases. We recommend extracting poppy DNA from 0.
The sample quality was more advantageous in the Qiagen extraction method; however, it is also significantly more labor intensive and, when considering the ratio standard deviations, the two methods perform similarly.
The DNA isolation method developed for latex is similar to the method developed for opium. Latex samples are commonly resuspended in water for long-term storage; therefore, the opium extraction method was modified to include the concentration of biological material. Latex samples had a lower DNA yield and low quality values Table 8. Like heroin, we expect this workflow to yield high amounts of amplifiable template DNA.
One gram of samples EE uncooked and OpC-1 cooked were extracted with the Qiastool kit; InhibitEX buffer and lysis reaction volumes were doubled to ensure samples were properly lysed and washed. Quantitative PCR was performed using the 2-stage amplification and the N primer set. The results indicate that DNA originating from both cooked and uncooked opium have been successfully extracted using the Qiastool kit and amplified using the 2-stage amplification method Fig.
In contrast, the cooked opium OpC-1 had a low yield exhibiting a Cp of Although both cooked and uncooked opium were successfully amplified, the DNA yield of the cooked opium may be improved through the extraction of higher amounts of starting material; therefore, the current optimal mass of starting material for extractions from cooked opium will be 1.
The N, N and N loci contain microsatellite sequences; therefore it is possible that size polymorphisms exist within the two genomic copies of the DNA in the diploid genome of a poppy plant.
Opium sample EE-2 amplified with N and N yielded a single primary allelic peak homozygote , with an amplicon length of The amplification using primer N yielded two primary allelic peaks heterozygote with amplicon sizes of and bases.
The resulting electropherograms also exhibit minimal artifactual noise, such as non-specific allelic peaks caused by electrical spikes or —A peaks caused by the non-template dependent terminal adenylyl-transferase activity inherent to many DNA polymerases, including the Roche Fast Start High Fidelity DNA polymerase. The stutter products correspond to products that are one or two repeat units smaller than the allelic peak. For example, the N locus contains a tetra-nucleotide repeat default n which, when amplified, resulted in the expected allelic peak at bases peak height rfu and a minor stutter peak at bases peak height: rfu.
The results demonstrate that Promega-isolated opium samples are of sufficient quality and quantity to yield amplifiable DNA and interpretable results. Select electropherograms obtained from the PCR amplification and subsequent CE-analysis of raw opium samples top-primer N, middle-primer N, bottom-primer N X-axis is fragment size in bases and Y-axis is relative fluorescent units. Note, a free-dye artifact is present at approximately bases; this is independent of the sample and can be disregarded from analyses.
PCR conditions were optimized in anticipation of the low quality and quantity of DNA encountered in processed opium and heroin samples. The EE opium sample was anticipated to exhibit elevated levels of inhibition and low DNA quantities due to characteristics such as moisture level, color and texture Fig.
The combined enzyme master mix with a PCR buffer pH of 9. The leaf sample displayed consistent amplification C p within each assay group. This was expected and served as a benchmark for the lower quality EE opium sample. To the best of our knowledge, this paper is the first to demonstrate successful DNA extraction of opium poppy DNA from heroin samples. The primary challenges were the low quality and quantities of DNA expected in processed opium and heroin samples coupled with the challenges posed from sample matrix issues and cutting agents.
In that regard, the successful extraction of DNA fragments is very significant. The quantities, as measured through the qPCR crossing point values, were low; however, this was not unexpected. The most critical finding was that we were able clearly distinguish opium poppy amplification plots from negative controls and background noise. Preliminary data indicate that we have successfully sequenced poppy DNA for some of these samples data not shown.
The opium gum or latex contains a mixture of alkaloids, terpenoids, phenols, and various proteins Opium is simply the dried latex that is collected from the opium poppy. It is typically in the raw form only dried or in the cooked form where raw opium is boiled, filtered to remove impurities, and reconstituted for heroin production.
The development of a successful DNA isolation strategy relies heavily on the ability to address the complexities present in latex or opium gum samples. Despite the increased quality metrics, the Qiagen procedure remains more labor intensive and less reliable compared to the automated Promega methods. Despite this, we recommend the use of the Qiagen stool kit as it proved to be more versatile. In order to improve the DNA yield and overall quality of heroin samples further, we employed a 2-stage amplification reaction using two DNA polymerases.
This dual polymerase system attempts to balance the amplification of damaged DNA while maintaining a high fidelity and minimize errors. This method led to more consistent responses than either polymerase alone. The combined effects of exceedingly low levels of damaged template DNA and the presence of inhibitors necessitated the modification of conventional analytical methods to increase sensitivity, for example, the cycle, 2-stage amplification mentioned above. The combined use of these methods resulted in the successful detection of opium poppy DNA from heroin and opium samples, but it also may have contributed to the low-level activity observed in the no-template controls.
The most commonly detected non-specific signals are due to the formation of primer dimers. The primer concentration can be lowered to avoid dimerization.
In addition, the use of such a high concentration is likely unnecessary when amplifying low-copy samples like those obtained from heroin extracts. The activity of the NTC is on average 4. The heroin-positive samples exhibited T m s distinct from the activity detected in the NTC with clear differences in curve morphology Supplementary Information Fig.
In addition, the signal obtained from NTC samples are reproducible across amplification reactions and instrument runs. We conclude that the NTC activity is not responsible for the primary signal obtained from the heroin positive samples.
While we believe that our proposed methods perform sufficiently well with high purity heroin samples i. Our inventory of opium and heroin samples included over samples consisting of white and brown powder and black tar heroin, as well as several types of opium. However, these samples were also limited in quantity due to the nature of the seizures in source countries.
In addition, few samples had ample mass to enable replicative comparisons of the methods we evaluated in this study. This limited the depth of our sub-studies and we recommend that the data presented herein be used as a starting point for further refinement of the procedure to extract opium poppy DNA from heroin.
For example, our small whole genome amplification sub-study indicated that there was no benefit to the use of these methods. Due to the nature and type of samples, any research group would face similar limitations to successful identification of poppy DNA caused by sample availability. The ability to obtain an opium poppy DNA profile from heroin opens a host of new avenues for law enforcement. We agree with the Journal of Pharmacy that it must be impossible to obtain a. Robertson, the successful producer in Virginia, states that his experience is very limited, he having only cultivated the poppy in a garden on very rich soil, where the yield of opium was very great; he neither measured the land nor weighed the opium.
He is satisfied that a deep rich soil is essential to a large yield; the poppy has a long tap root, which enables it to stand severe drought, provided the tap root can penetrate the soil to a sufficient depth. He thinks alluvial soils are best. The young plant is very tender, of slow growth, and cannot be successfully transplanted. The seed should be put in drills about three feet wide, the plants standing from one foot to eighteen inches apart, or even more, as it is a very vigorous grower.
The last of July or early in August is a good time to sow the seed, as the plants stand the winter without injury. The single poppy he found to yield more opium than the double, and there is less trouble in obtaining it from the capsules.
The single white poppy, or rather the poppy, with white seeds, is generally considered the true opium plant. When the capsules are about half grown, or three or four days after the flower has dropped, is the proper time to make several longitudinal incisi ,ns on the capsule, taking care not to cut through the capsule.
The incisio m should be made during the latter part of the day, and the thickened juice which exudes during the night scraped off the next morning with a dull knife. A few erroneous analyses of some plants of the poppy family were made in the early days, but these were later corrected. It is probable that Papaver setigerum -the closest relative of Papaver somniferum -does produce morphine, but no record of a careful botano-chemical study was found.
In any case, Papaver setigerum is only a rather rare wild flower, found in the Mediterranean region, occasionally grown in gardens but not grown commercially even for floral purposes. Other poppies, such as the California poppy Eschscholzia californica , the Mexican poppy Argemone mexicana , the Oriental poppy Papaver orientale , and the common "corn poppy" or "field poppy" of Europe, the progenitor of the Shirley poppies Papaver rhoeas , have been analysed and other kinds of alkaloids found in them, but not morphine.
By original work it was shown that the Tulip poppy Papaver glaucum , though listed as a close relative of Papaver somniferum , is chemically more closely related to Papaver rhoeas, and contains no morphine.
In the beginning the Narcotics Bureau had proceeded largely by suasion, because there was not a firm legal basis for more vigorous action. Even after the Opium Poppy Control Act came into effect, the California farmers had been allowed to harvest their poppy-seed, because cultivation had been started before the law was enacted, and the violations were not wilful. Now the violations were wilful-though supported, to a degree, by some state officials-and it was evident that effective action must be taken or the law acknowledged to be a complete failure.
Beginning early in January , registered letters began to go out to the growers, warning them that they were in violation of the Federal law, regardless of state permits. Some ploughed up their poppy-fields and planted something else; but others replied with form letters claiming that they had a right to produce poppyseed.
They were banding together to fight the law. The growers now left were chiefly well-to-do men who were willing to gamble on having their poppy crop destroyed by the Government, in the hope of enormous profits in case they were able, to bluff out the Narcotics Bureau, or defeat the law in a court case.
They insisted on their "right" to produce a food crop regardless of narcotic dangers. What they refused to take into consideration was the fact that the reason the price of poppy-seed remained so high was that all other farmers were obeying the law-while they proposed to profit immensely by disobeying it. Early in March , the Commissioner of Narcotics recommended that the poppy plants in several fields be destroyed by narcotic agents, as a test case. Largely because of the state permits, it was decided to act more slowly, through the Department of Justice.
In this month the Secretary of the Treasury found it necessary to say, in reply to poppy propaganda sent to the Vice-President:. This is incorrect. We have caused analyses to be made by both government and private chemists and have abundant proof that the blue poppies grown in California are opium poppies.
If you insist on growing poppies this year, I shall be compelled to report you to the United States Department of Justice for prosecution". At the same time the Commissioner issued instructions that an agent be sent to inspect the fields. The narcotic inspector who was sent reported, on 1 April , that thirty-three fields aggregating 1, acres had been planted. A few to whom permits had been issued had not planted, and one grower had ploughed his plants under on about 16 March.
Even after the several years of controversy, it was found that two or three farmers were growing poppies without state permits; they had applied for permits but had gone ahead without having received them.
A narcotic agent was sent to join the inspector; the two were to keep continuous watch over the situation until it was finally settled. The narcotic "agents" usually worked on enforcement cases, while the "inspectors" chiefly supervised the legal sales and use of narcotics.
By the beginning of May, the growers had received their letters from the Commissioner of Narcotics and a number of them had decided to give up On 4 May, the two narcotics officers reported as follows: Poppy fields ploughed under, 10; in process of disking, 5; did not plant, 3; verbal promise to disk, 3.
On 5 May, they reported that twenty-five growers had not yet ploughed under their fields, but ten of them were about to do so, leaving fifteen growers who had acres in poppies. The earliest analysis of the current poppy crop. A land-owning company wrote to the Commissioner reporting that a farmer leasing some of its land was growing poppies; the officials of the company inquired about their own responsibilities in such cases. The District Supervisor of Narcotics was directed to obtain samples and find out if these were opium poppies.
This he did although he found that the cultivation was under state permit and the plants, at the time, were very small-approximately two inches in height and having only 2 to 4 leaves.
Not much was known about the morphine content of such very small plants but it was thought to be minute, consequently the chemist asked for a sample of five pounds. He got two pounds, and to secure this amount almost an eighth of an acre was denuded of plants.
He had no difficulty in proving the presence of morphine in this material. As soon as it appeared that some cases would be fought in court, the narcotic officers began systematically gathering samples in triplicate. They were sent to three government chemists who were experienced in narcotics work; one in San Francisco, California, one in St. Paul, Minnesota, and one in Washington, D. At first, whole young plants were pulled up; later the green capsules were cut off.
No attempt was made to make the three samples identical, but in each case three samples were taken from the same field at the same time. There were no fundamental differences in the methods of analysis, nor in the results, though it will be noted that one set of results was consistently a little higher than the other on the young plants.
The agreement of analyses for capsules from the same field was good. Many of these capsules were quite immature and none had reached full maturity and ripeness.
This probably accounts for the fact that none of them. About 0. In examining the dried plant material it was discovered that alcohol would extract only about half the morphine. This was unexpected, as some methods of examining plant material for alkaloids call for extraction of the powdered, dried material with alcohol. It was found that alcohol could be used to take out all the morphine from fresh, green material, but in this case, of course, the moisture of the leaves was also abstracted, presumably carrying the rest of the morphine with it.
A satisfactory procedure for the dried material was found to be continuous extraction with warm or hot water for a number of hours. It was found that the capsules contained about nine-tenths of the average United States Pharmacopoeia dose of morphine per capsule.
This was an understandable way of expressing the content regardless of whether the capsules were green or dried. In several of the most important cases, the green pods, shipped by air express to Saint Paul, would still yield the milky juice when incised. Although no more than a few centigrams of opium could be collected in each case, the chemist wished to complete the proof as to the identity of these "opium poppies".
The droplets of juice were collected on one of a pair of balanced weighing glasses "watch glasses" and dried at 60 degrees centigrade. The very accurately weighed opium was then assayed by extraction methods, the separated morphine being titrated with hundredth normal acid.
The morphine percentages on the dry opium in four different cases were: In the last case it was also determined that 4. In Canada, in the spring of , another case of abusive use of poppy capsules was discovered and prosecuted. In California the poppy plants grew with great rapidity during April Early in May, the District Supervisor conferred with the District Attorney on a procedure to seize the crops. On 10 May , one of the California Congressmen asked that action be deferred, but the Commissioner of Narcotics replied, "Arrangements have been made to proceed under the law to forfeit the crops".
At the same time he sent instructions to his District Supervisor to begin destroying poppies at once. On 11 May, the latter objected that the United States Attorney had proposed a different procedure.
We want these crops taken out of circulation, either through the District Attorney's method, or through our own efforts You have the entire responsibility from now on whether to follow the District Attorney or to plow under, whichever is going to be effective.
Both or either have our approval". In the meantime the District Attorney had prepared papers for seizing the poppy crops. At the same time he sent a final registered letter, to each grower:. Law", another commented editorially on the "Opium Rebellion" and condemned the "recalcitrant farmers".
At this time 17 May the score stood:. Most of the remaining growers would not budge, and the District Supervisor wrote asking for more explicit written instructions. On 19 May he was given explicit instructions by teletype:. You are instructed personally to arrest and file complaints against all growers who have not destroyed poppies". The District Supervisor then went to the poppy locality-Chico and vicinity-and on 22 May personally arrested seven growers. Three of them promptly retaliated, through their attorneys, by filing a complaint against the Bureau of Narcotics, the Commissioner of Narcotics, the United States Attorney, the District Supervisor of Narcotics, and the narcotic officers who had been active, alleging that the Opium Poppy Control Act violated the Constitution of the United States.
The newspaper headlines 23 May read as follows:. Notices of seizure were posted for acres of poppies. Apparently the return is somewhat exaggerated: in the average yield was pounds per acre, but in the best fields it went to pounds. Some of the growers now decided to destroy their crops, but on 27 May three of them changed their minds and joined the others, in spite of the fact that they had already destroyed a considerable part of their crops.
Only those were to be indicted who had not ploughed up, disked in, or destroyed their poppies. Nine growers were indicted in seven distinct cases. It was not the Government's case against them which came to trial, however, but the complaint of six of the growers and their appeal for an injunction against the government officers. The Tenth Amendment, by invading the reserved right of the State of California to provide for and regulate the production, cultivation, and growing of agricultural crops within its borders, When the growing and production were conducted solely for intrastate transactions;.
They also alleged that the officers were endeavouring to apply the Act in such a way as to deprive the plaintiffs of their property without just compensation, and that the enforcement of the Act did not come within the police powers of the United States so far as the growing and production of edible blue poppy-seed was concerned.
That it be decreed that edible blue poppies raised solely for poppy seeds are not an opium poppy, nor a plant which is the source of opium or opium products,and that the production of edible blue poppies does nor come within the purport or application of the Opium Poppy Control Act of ;. Welsh granted a temporary restraining order against the Government, to protect the crops from destruction until the case could be heard. Although the Press generally favoured the Narcotics Bureau, the farmers who had been growing poppies or wanted to do so had considerable influence.
In addition, some state officials, especially of the state Department of Agriculture and the Attorney-General's office, had, from the start, put the state in the position, of favouring poppy cultivation. Under these influences the state legislature made a move which was doubtless intended to help the poppy growers in their case, though actually it could not legally affect what they had already done contrary to existing law, Headlined "Poppy Growing Approved by State Legislature", the news item of 8 June read: "An unanimous vote in the assembly today completed the adoption of a joint resolution by Senators Gordon, Crittenden, and Denel, asking Congress to enact legislation recognizing the importance of blue poppy seed for food uses and permitting its production".
Because of its urgency, it was decided that the case should be heard by a Statutory Emergency Court, consisting of two District Judges, Martin I. Welsh and Louis R. This procedure was adopted in order to get action, promptly, in regard to the existing poppyseed crop-which was ready for the harvest-and to dear the way for a final decision on the constitutionality of the Opium Poppy Control Act. The three-judge Court decided to make its decision primarily from briefs and affidavits, without requiring oral testimony of witnesses.
In the meantime, several hearings were held before the District Judge. On 19 June, the growers made a motion asking that they be permitted to harvest their crop. This was denied. On 7 July they filed a new motion to the same effect. At the hearing on 10 July it was shown that three of the growers had mowed some of their poppies anyway, preparatory to clearing the ground for other crops.
The18 poppy plants were, however, apparently just left lying in the fields at this time. The motion of the growers was again denied and they were strictly ordered not to touch any of the poppies in the field again, until the Court had made a decision.
On 27 July, the attorney for the growers suggested that the crop be harvested and sold and the proceeds impounded, and in the event the litigation resulted unfavourably to the growers that the entire proceeds be turned over to the American Red Cross. But this offer came too late and was unacceptable anyway, because, for two years past, the recalcitrants had been harvesting poppy-seeds, while those more willing to obey the law, or the wishes of the Narcotics Bureau, had destroyed their plants or given up the cultivation.
Extravagant claims were made by the growers that their agricultural poppies produced only insignificant amounts of morphine in the capsules at a late stage of growth; that all poppies produce opium and morphine, including the California poppy Eschscholzia californica , the state flower of California; and that innumerable other agricultural plants produce poisons in some part of the plant or at some stage of growth, so that there was no more reason to prohibit the cultivation of poppies for food purposes than of tomatoes, potatoes, lima beans, rhubarb, lettuce, tapioca, apricots, and cherries!
The analytical results of course showed plainly the amount of morphine in the poppies under attack. To further combat the claims of the growers, an affidavit was prepared by one of the government chemists, part of which will be quoted here:.
The basic question, of course, was simply whether or not the Opium Poppy Control Act was constitutional: there was no question but that the poppy growers were violating it. There is no need to quote the brief for the "defendants"-the United States Attorney and the District Supervisor of Narcotics-as the views of the government attorneys were substantially accepted by the Court.
On 28 August , the Court handed down its decision unanimously affirming the constitutionality of the Opium Poppy Control Act.
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