Sowing takes place in the autumn, after the first autumn rains moisten the earth. If these rains fall too late, the plant is insufficiently developed by the winter, and is therefore sensitive to cold and may be destroyed even in a mild winter.
Another risk of crop failure is that of a hard winter, especially without snow. An unduly mild winter is also unsuitable, for the plants can develop too fast and the opium gathering period may occur too early, when rains are more frequent. A wet spring can promote the development of plant diseases to which the poppy is very sensitive and too much moisture in the ground makes the latex too thin and causes it to trickle out of the capsule on to the ground after incision.
The chief danger of the producer, however, is rain at the time of the harvest, as the harvest can be potsponed for only a few days. Rain during the night following the incising of the capsules may entirely wash away the drops of latex and completely destroy the crop. Even dew can cause serious damage at that period.
In the case of spring cultivation, there is the further risk of dry weather setting in before the plants are sufficiently developed, so that they may wither without yielding either opium or seeds. The opium poppy is one of the most intensive agricultural cultivations and requires a great deal of manpower; the poppy needs constant care throughout its period of growth.
Approximately 30 days of team work and manpower days including collection of opium and seeds are required to cultivate one hectare. A shortage of specialized labour at harvest-time is yet another risk to which the cultivation of the opium poppy is exposed.
The hazards of opium poppy cultivation may best be seen from table 1. While 70, kg were produced in , only 2, kg were produced in The mechanization of agriculture cannot make the production of opium much cheaper. Apart from ploughing, sowing and winnowing the seeds with ploughs, seeders and winnowers the remaining operations can be performed only by human hand. Labour takes up from 80 to 90 per cent of the production costs. From above it is evident that opium is not suitable for large state or co-operative farms, using modern agricultural techniques, nor for large areas in general.
However, the opium poppy is being cultivated also on state and co-operative farms, but over, relatively small areas, as well as on individual farms. From to , the following areas of Yugoslavia were sown with poppy average areas are given.
General provisions on control over the production, manufacture and processing of and trade in narcotic drugs are contained in the Act on Narcotic Drugs of 23 January Raw opium may be produced only in authorized areas.
Every year, each producing district announces the total areas sown with opium poppy, on the basis of production authorization issued to the producers. In any given year, the producers are bound to turn over the total amount of raw opium they have produced to the "Bilka" enterprise at Skopje, which alone is authorized to purchase it.
Any producer who does not do so or who sells raw opium to unauthorized persons is liable to criminal prosecution. Control over sown areas and the quantities of raw opium put up for sale is effected by the local authorities, who, surveying the sown areas, can also estimate the amount of opium obtained, although it varies widely from one district to another according to weather conditions.
The fact that a large number of people, mostly neighbours and local inhabitants, must take part in incising the capsules and collecting the opium makes it much more difficult to conceal the opium obtained. Every year before sowing, the producers may make an agreement with "Bilka" in advance, for an interest-free loan in fertilizers or in cash, on condition that the loan will be converted when the crop is purchased.
Moreover, the purchase price is guaranteed in advance for the producers, expressed in morphine units. This compulsory purchase is effected at different times in different places. The day when opium is to be purchased at a given place is announced in advance. The purchase is carried out by a commission consisting of three members, one of them an expert of "Bilka" and another a representative of the local authorities.
Should the judgement of the producer and of the commission or one member of the commission concerning the quality of the opium morphine content fail to coincide, either side is entitled to ask that a chemical analysis should be made. The cost of the analysis is borne by the side whose appraisal is proved to be wrong. Experts can estimate the morphine content with reasonable accuracy. Experience has shown, however, that even they can make considerable errors; no estimate whatsoever can be regarded as absolutely reliable.
All commercial firms and factories authorized to deal in narcotic drugs, as well as pharmacies and hospitals, have to keep special registers of their trade in narcotic drugs. Delivery vouchers, doctors' prescriptions and hospital vouchers for hospitals and pharmacies serve as the documents on the basis of which the register of narcotic drugs is kept. The register has numbered pages, and is controlled and stamped by the authorities, who affix the control ribbon and seal which make it impossible to alter the entries.
The control system is greatly facilitated by the fact that there are no private trade enterprises or factories in Yugoslavia and no private pharmacies and health institutions. Within the Federal Administration for Pharmaceutical Service and Medical Supplies there is a Commission on Narcotic Drugs, consisting of the representatives of federal institutions interested in the problem of opium and narcotic drugs.
The Commission's task is to co-ordinate the work of the interested departments by enforcing the provisions of the Act on Narcotic Drugs, applying laws and regulations on narcotic drugs and carrying out the obligations arising from conventions and other international instruments.
Within the Committee for Foreign Trade there is an Opium Office which controls the import and export of narcotic drugs and issues import and export licences. Yugoslav opium is put on the market in the form of hard bricks, 2 to 2.
Each brick bears the print of a thrice-incised poppy capsule and the inscription "Bilka ", Jugoslavija see figure 5. The opium is granulous when broken, dark reddish-brown in colour, with a characteristic smell and an acrid, unpleasant and very bitter taste.
Under the microscope very few plant particles can be seen particles of capsule and leaf and under the polarizing microscope a preponderance of small crystals, with a few square grains and sometimes a few rods are visible. Yugoslav opium has the highest morphine content in the world. The morphine content calculated in dehydrated opium is more or less constant and varies between The most characteristic property which distinguishes Yugoslav opium from other opiums is its typical ultra-violet absorption spectrum.
According to the ratio between these two alkaloids, Yugoslav export Macedonian opium, unlike the majority of the Asian types, distinctly falls into the papaverine type. Similar results to a certain extent are obtained in the case of Greek opium and some Turkish types.
It is interesting to note that the opium which has been grown on an experimental basis in northern Croatia is quite different [ 16 ] and belongs to the thebaine type. Another characteristic is the composition of the ash content. It has also been found that the quantity of yellow pigment in Yugoslav opium increases constantly and steadily as it grows older, a fact which can be used as an approximate assessment of its age.
In recent years, poppy capsules together with poppy straw have gradually been replacing opium as raw materials for the production of alkaloids. Dried and crushed capsules are mainly used, together with approximately 10 cm of the stem under the capsule. In suitable soil, the highest morphine content is found in ripe capsules, while in poor soil it is found in capsules which are not quite ripe. Accordingly, in the period before the Second World War, a clear distinction could be made between two categories of producer.
One of these categories consisted of town dwellers contractors, landowners, tradesmen, traders, etc. When labour was relatively cheap, this form of production was in most cases profitable. However, in periods when the price of opium on the world market fell considerably, these producers used to abandon opium cultivation and turn to other profitable but less risky crops. Thus, for example, the vertiginous fall in prices in and between January and December , the world prices of opium fell to one-sixth , led to a decrease in the area sown from 14, hectares and 10, hectares to 4, hectares in After this crash, the producers' confidence in this crop returned very slowly, which will be seen from the gradual increase of sown areas to some 5,, 6, and 8, hectares in the following five years see table 1.
The second category of producers consisted and still consists of rural small-holders, owning very small plots of land worked by their own labour and that of members of their household. Since these small undertakings do not use or use very little hired labour, their production costs are quite differently distributed.
The cost of labour does not mean much to them: it is far more important to be able to use household labour at a time when there are usually no other operations to be performed on the land. Moreover, such farmers are often tied to opium poppy cultivation by the fact that their land would yield much less of any other type of crop.
These are producers who therefore remain faithful to the opium poppy and on whom fluctuations of world prices have considerably less effect. The large-scale public works which were undertaken in the People's Republic of Macedonia in the post-war years hydro-electric power stations, roads, factories , the industrialization of the country, mechanization and certain technical measures in agriculture have resulted in a gradual rise in the standard of living and consequently in a rise in the cost of labour.
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This study is the first to report the successful development of a method to extract opium poppy Papaver somniferum L. DNA from heroin samples. Determining of the source of an unknown heroin sample forensic geosourcing is vital to informing domestic and foreign policy related to counter-narcoterrorism. Current profiling methods focus on identifying process-related chemical impurities found in heroin samples.
This study focuses on methods to optimize the DNA extraction and amplification of samples with low levels of degraded DNA and inhibiting compounds such as heroin. We compared modified commercial-off-the-shelf extraction methods such as the Qiagen Plant, Stool and the Promega Maxwell RNA-LEV tissue kits for the ability to extract opium poppy DNA from latex, raw and cooked opium, white and brown powder heroin and black tar heroin.
Opium poppy DNA was successfully detected in all poppy-derived samples, including heroin. The modified Qiagen stool method with post-extraction purification and a two-stage, dual DNA polymerase amplification procedure resulted in the highest DNA yield and minimized inhibition. This paper describes the initial phase in establishing a DNA-based signature method to characterize heroin. The heroin epidemic has become a widespread domestic issue for the American people; it has touched the lives of families who have lost loved ones to heroin overdoses and is ubiquitous in the mainstream media.
The heroin epidemic shows no signs of slowing. The number of individuals using heroin approached one million by the end of , an increase from previous years 1. Investigative and intelligence information regularly compiled by such entities enable the U. In addition, the opium poppy cultivation and heroin production in various source countries are also being investigated. Opium poppy Papaver somniferum L. While the poppy straw harvested from cultivation is generally employed by pharmaceutical entities, the liquid or dried sap known as opium latex or gum that is obtained by lancing the outer surface of poppy pods is the starting material for the clandestine production of heroin.
The research into opium alkaloid chemistry began with the isolation of morphine in the early s. Several publications cite the presence of 80 or more alkaloids present in opium.
The alkaloid composition of opium can be varied because of agronomical, climatic, or cultivar differences. The U. The HSP involves four diverse signature methodologies that are independently employed 2 , 3 , 4 unpublished research at the laboratory on Isotope Ratio Mass Spectrometry. Despite the significance of poppy varieties and their unique alkaloid profiles in the HSP, there have not been efforts to develop a signature method using opium poppy DNA. The chemical processing steps involved in producing heroin from opium require extreme alkaline and acidic pHs and high temperatures As a result, heroin samples are expected to contain exceedingly limited amounts of damaged and degraded poppy DNA.
The lack of development efforts were likely due to the low likelihood of obtaining a suitable DNA signature. However, the genetic profiling of opium poppy remained a topic of great interest to stakeholders such as the Drug Enforcement Administration Special Testing and Research Laboratory 5 , 6 , 7 , 8 , 9 , 10 , Advancements in DNA amplification and sequencing technologies in the last decade prompted an innovative scientific investigation into utilizing the poppy DNA fragments detected in heroin as a classification tool.
The genetic characterization of Papaver Somniferum L. The genesis of this applied work relied on foundational genomic research that began characterizing the opium poppy. This has been central to the ability to exploit the opium poppy DNA for use in determining population-based genetic identity. The diploid nature of the poppy genome imparts a much simpler workflow for genetic identification, specifically because it mirrors the approaches used for human genetic identification using single copy DNA sequences.
The purpose of this study was to identify and characterize microsatellite nucleotide sequences based on repeat composition and abundance in the genome. It permitted further research that focused on various aspects of genetic identification of the opium poppy. Although these microsatellites are of potential use for geosourcing opium poppy, we needed to mine the genome to ensure the microsatellites and single nucleotide polymorphisms had significant levels of variation among the relevant populations to permit geosourcing.
This manuscript presents the first results attempting to employ opium poppy genetics for the classification of heroin. While the identification of variety-specific and geography-specific DNA markers continues as part of an elaborate scheme of poppy genome research, the optimization of a DNA extraction method from crudely manufactured black tar and highly refined powder heroin samples, and its subsequent amplification are presented here.
Isolation of Papaver somniferum L. DNA from heroin presents a new and increasingly complex challenge compared to that encountered in the extraction process for opium. Heroin samples are inherently diverse. They vary in purity, cutting agents, level of processing and storage conditions.
These factors have a significant impact on the resulting quality and quantity of opium poppy DNA obtained from the sample. 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.
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